J LIBRARY
. ; 1 I I '
ON SCHOOL OF MEDICINE FOR WOMEN,
Hunter Street, Brunswick Square, W.C.
Number
/
PHYSIOLOGICAL CHEMISTRY
OF
THE BRAIN.
WBWOMEH
A TKEATI
THE CHEMICAL CONSTITUTION
OF
THE BEAIK
BASED THROUGHOUT UPON ORIGINAL RESEARCHES,
BY
J. L. W. THUDLCHUM, M.D.
FELLOW OF THE ROYAL COLLEGE OF PHYSICTTfTS, LONDON ; PRESIDENT OF THE WEST
LONDON MEDICO-CHIRURGICAL SOCIETY, ETC., ETC.
LONDON :
BAILLlilRE, TINDALL, AND COX,
20, KING WILLIAM STEEET, STEAND.
' 1884.
■ t
TO THE
RIGHT HONOURABLE LORD SHERBROOKE,
OF SHERBROOKE,
GRATEFUL REMEMBRANCP] OF THE HIGH-MINDED LIBERALITY WITH WHICH,
DURING HIS TENURE OF THE OFFICE OF VICE-PRESIDENT OF THE COUNCIL,
HE AIDED THE ORIGINAL INSTITUTION OF THESE RESEARCHES, AND
OF THE ENLIGHTENED COUNTENANCE AND SUPPORT WHICH HE
HAS CONTINUED TO GIVE THEM DURING THEIR
PROGRESS,
BY
THE AUTHOR.
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PEEFACE.
A CENTURY has nearly elapsed since the brain was for the first
time made the object of physiological research, with the aid of
the then newly discovered science of chemistry. But the attempt
remained isolated at the time, and was renewed only at long
intervals, mostly by inquirers who had no cohesion and little
sympathy with each other, and particularly had no paramount
object in the prosecution of such an inquiry. Now and then an
eminent chemist just touched the problem, to leave it soon after
he had recognised its inherent difficulties. During the last twenty
years only were more frequent attempts made to approach the
knowledge of the chemistry of the brain ; but these results were
so little satisfactory, that inquirers could not even think of pro-
ceeding to quantitative investigation of normal relations, much
less of studying pathological phenomena. It was under such un-
promising conditions that I undertook, about twelve j^ears ago,
to make some researches on this subject for the then Medical
Department of the Privy Council. The j)rogress which I was
able to report in 1874 was sufficiently great to ensure acquiescence
in the continuance of my efforts on the part of the former authority
and subsequently of the Local Government Board, to whom the
functions with regard to the administration of the Parliamentary
grant for researches in aid of pathology and medicine, previously
exercised by the Privy Council, were transferred. It soon became
evident that the physiological inquiries, which had been intended
as an introduction to pathological ones, would occupy far more
time than had been anticipated. However, as the information
grew, as the discoveries multiplied in number and gained in pre-
cision, no hesitation could be felt in following up the researches
to a decided issue. Thus I came to trace the foundations of the
viii
PE E FACE.
chemical constitution of the brain, the outlines of which I have
the honour of laying before the medical profession and the general
scientific public in the shape of the present treatise. The work
is a systematic consolidation of all the researches on the subject
which have been laid before Parliament in the Annual Eeports of
the Medical Officer of the Privy Council and Local Government
Board respectively. The matter of the work is therefore public
property, and under these circumstances I think it my duty to
specially avow my responsibility for its contents.
I should not be surprised if some readers were, at the first
glance, to think the subject recondite and its treatment heavy.
Descriptions of complicated chemical processes are necessarily
tedious to those who have no practical concern in their repetition
or even knowledge. By some they will j^robably be treated with
the consideration which Hellenic literature received before the
Eenaissance, and which has been recorded in the sentence : ' Grseca
sunt, non leguntur.' But I would beg all readers to take into
patient consideration that I had to write a highly technical work
on a most difficult subject, and to endeavour to make it accept-
able, in such a form as it practically could have, to anatomists,
physiologists, and ^pathologists, as well as to that principal part of
the medical public which is engaged, like myself, in the practical
pursuit of the profession.
However, to all readers who will enter upon the consideration
of the subject with the intention of adding it to their previous
stock of knowledge, I can promise the enjoyment of some intel-
lectual pleasure. They will find the brain to be the most diver-
sified chemical laboratory of the animal economy; they will find
such numbers and varieties of hitherto unknown and most remark-
able chemical principles taking part in such complicated chemical
structures and processes, that the explanation of the mental
phenomena, and of their aberrations under the influence of
disease, seems much less difficult than it appeared before these
discoveries.
I have not in this treatise entered upon any pathological con-
siderations, although I have in several smaller publications shown
the bearings which some of my discoveries have upon the practical
study and treatment of diseases. Thus I have shown the morbid
alteration of the nerve-marrow in locomotor ataxia, and the
occurrence of a kind of glycohfemia to be intimately connected
BREFACE.
ix
with patholytic changes in substances of the group of cerebrosides.
I could go further and unfold, e.g., a chemical connection between
the function of the liver and that of the brain, opening views into
the pathology of the future and illuminating, though only with
the disappointing brevity of an electric spark, regions as dark as
those of general paralysis and melancholy. But I have (in early
stages of my work) formed the resolution never to propound a
generalisation on any subject before having proved the validity
of all data obtainable by observation or experiment. And I must
say that I have not yet found any subject in chemical biology
on which, governed by that resolution, I should have liked to
proceed to generalisation.
The principal reason for this abstention is the circumstance that
the data or so-called facts available in chemical biology are as yet
too incomplete, and therefore unsuitable for connected treatment
except with the aid of hypotheses. This circumstance also affects
the present treatise, and I have taken care to point out to the
reader cases where my information was partial, or where no data
at all were as yet available. In order, however, that the reader
may not, from this avowal, come to an erroneous conclusion
regarding my own estimate of the value of the researches com-
municated in this treatise, I undertake to assure him that they
are of fundamental importance, and that all further developments
in chemical neurology must start from them as a basis. I say this
in view of the records of the work of all those who have grappled
with the problem before me, and in kindness to all who may like
to deal with it hereafter.
The literary discussions on subjects of brain chemistry which
have taken place during the last few years, and in which I was
obliged to take an active part, have had such an issue that I was
happily enabled to exclude all controversy from the pages of my
treatise. I have moreover endeavoured throughout to confine
my matter to the narrowest space compatible with clearness of
description and accuracy of demonstration, and it is owing to this
resolution that I have omitted not only references to historical
literature, but multifarious details of analytical figures, the repro-
duction of which would have enlarged the size and enhanced the
price of the work without any corresponding advantage to the
greater number of readers. Those, however, who value such
matters will find a complete history of research in brain chemistry
xii
PREFACE.
detriment of the science of which, even by a short connection,
their names have become permanent ornaments.
I am in hopes that several of the discoveries made on the brain,
and communicated in this treatise, will be able to shed some light
upon other subjects in biological chemistry, which are at present
little understood. I have indicated some of my ideas on that
subject in a few pages appended to the division on cerebral
phosphatides, which give a short, comparative consideration of
other phosphatides of the animal body.
Phosphatides are the centre, life, and chemical soul of all
bioplasm whatsoever, that of j^hants as well as animals. Their
chemical stability is greatly due to the fact that their fundamental
radicle is a mineral acid of strong and manifold dynamicities.
Their varied functions are the result of the collusion of radicles
of strongly contrasting properties. Their physical properties are,
viewed from a teleological point of standing, eminently adapted
to their functions. Amongst these properties none are more
deserving of further inquiry than those which may be described
as their power of colloidation. AYithout this power no brain as
an organ would be possible, as indeed the existence of all bioplasm
is dependent on the colloid state.
I was preaching in the desert when, in 1866, I advanced the
hypothesis, based upon numerous and deep investigations, that
the fall of temperature in the collapse-stage of cholera, far below
the normal, is due to the destruction of the colloid state in
myoplasm, produced by the direct influence of the disease-cause.
Now, in 1884, we can demonstrate to the eyes that there are
bacteria or microzymes which have the power of liquifying
colloids, while there are others which have not got that power.
That the phosphatides may be capable of being affected by the
former is very probable, particularly when it is observed how in
cells bacteria congregate and multiply in close proximity to the
nuclei. Now so-called softening of the brain consists in the first
place merely in the loss of the colloid state on the part of a smaller
or larger portion of nerve-marrow. The bacilli of tubercle may
effect such a softening ; but others may have the same power.
The next stage after the fluidification is patholysis, the splitting
up of the liberated immediate principles into their proximate
nuclei, the same as those which we obtain by chemolysis. The
two processes, the one in cholera, and the other in softening of
PREFACE.
xiii
the brain, illustrate the acute and chronic form of loss of colloida-
tion and of patholysis under the influence of microzymes.
Some phosphatides of the brain probably permeate the neuro-
plasm in a non-colloid dissolved liquid state, e.g.^ amidomyelin ;
and this body has the remarkable property that while perfectly
soluble in water, and non-colloid at the ordinary temperature and
at temperatures approaching that of the animal body, it becomes
colloid at temperatures between the normal and the highest fever
heat. It is not impossible that some such change may be the
cause of death in many febrile conditions, and in many cases of
death from exposure to excessive heat, in which no adequate
mechanical lesion can be discovered.
A few further examples may suffice to indicate some of the
lines on which the practical consideration of diseases of the brain
by the aid of its chemistry will, at least in the first instance, have
to proceed. Many kinds of headache are probably due to intra-
cranially brewed chemical poisons, or to poisons carried from the
body to the brain by the blood, whether fermented in the body,
or like alcohol, morphia, and fusel oil, formed out of the body.
From such occasionally produced effects to the constant produc-
tion of similar effects by a continued zymosis, be it now caused
by organised or unorganised ferments, is not a great step. Many
forms of insanity are unquestionably the external manifestations
of the effects upon the brain-substance of poisons fermented within
the body, just as. the mental aberrations accompanying chronic
alcoholic intoxication are the accumulated effects of a relatively
simple poison fermented out of the body. These poisons we
shall, I have no doubt, be able to isolate after we know the
normal chemistry to its uttermost detail. And then will come in
their turn the crowning discoveries to which all our efforts must
ultimately be directed, namety, the discoveries of the antidotes to
the poisons, and to the fermenting causes and processes which
produce them.
THE AUTHOR
11, Pembroke Gardens, W.,
February, 1884.
J
CONTENTS.
I.
INTRODUCTIOK
General view of the subject matter . , . . .1
II.
METHOD PUESUED FOR THE ISOLATION OF IMMEDIATE
PRINCIPLES.
PAGE
Preparation and comminution of braintissue . . .28
Extraction of brainpulp and separation of albuminous or insoluble
matter, white matter, buttery matter, last oily matter, and
ultimate watery mother-liquor . . . . .29
Process for separating white matter into its constituents . .32
Treatment of the buttery matter . . - . . .34
Treatment of the last oily matter . . . . .35
Summary of immediate principles in the crude state or mixtures
thereof isolated . . . . . . .35
Summary and arrangement in groups of immediate principles . 36
IIL
GROUP OF PHOSPHORISED PRINCIPLES OR PHOSPHATIDES.
General properties of the phosphorised principles . . .39
Deposition of impurities . . . . . .40
Filtration of the watery solutions of the phosphorised matters . 41
Solubility of phosphorised matters in ether and alcohol . .42
A. Subgroup of Mononitrogenised Monophosphatides. N : P = 1 : 1 . 42
1. Lecithins . . . . . . . .42
Definition . . . . . . . .42
Isolation . . . . . . . . 43
Episode concerning the shifting of cadmium chloride in mixtures of
lecithin, parainyelin, and amidomyelin during recrystallisation
from spirit . . . . . . .44
Continuation of the description of the process • . . .45
Separation of lecithin from amidomyelin and paramyelin when all
are in the free state . . . . . .45
Properties and compounds of lecithin . . . .46
xvi
CONTENTS.
PAGE
Lecithin cadmium chloride . . . . . .48
Chemolysis of lecithin . . . . . .49
Note on oleic acid and its reaction with oil of vitriol and sugar . 51
Theory of lecithins considered as phosphatides . . .52
2. Kephalins . . . . . . . .52
a. Kephalin . . . . . . .52
Purification . . . . . . .52
Expulsion of hydrochloric acid from kephalin by water . . 53
Bases and salts which are in combination with kephalin after
filtration of its aqueous solution . . . .54
Dialysis of kephalin , . . . . .55
Clarification and decolorisation of watery and ethereal solutions
of kephalin . . . . . . .55
Influence of animal charcoal on water solution of kephalin . 56
Bearing of kephalin in ether with charcoal . . ,56
Ultimate analysis of kephalin . . . . .57
Solubilities of kephalin . . . . . .58
Reactions of the aqueous solution of kephalin . . .59
Compounds of kephalin . , . . . .61
Kephalin cadmium chloride . . . . .61
Kephalin with hydrochloric acid and platinic chloride . . 62
h. Amidokephalin . . . . . . .64
c. Oxikephalin with cadmium chloride . . . .64
Behaviour of a similar salt with water . . . .66
(1. Peroxikephalin . . . . . . .66
Transformation of this body into lead salt . . .66
Comparison of the organic matter in the lead salt, with the com-
position of the free body and of the organic matter in a salt of
kephaloidin with cadmium chloride . . . .67
€. Kephaloidin . . . . . . .67
Definition . . . . . . .67
Solubility in water and filtration. . . . .68
Bearing in dialysing apparatus ; first and second experiments . 68
Bearing of the ether solution with water , . .68
Reactions of the watery solution of kephaloidin . . .69
Kephaloidin lead. . . . . . .69
f. Oxikephaloidin with cadmium chloride . . . .70
Comparison of the composition of the organic matter with other
kephalins . . . . . . .71
(f. Decompositions of kephalin . . . . .71
li. Chemolyses of kephalins . . . . .72
a. Limited chemolysis by caustic soda . . . .72
Reactions of the soapy solution . . . .73
Decomposition of the soaps b}^ hydrochloric acid ... .73
The precipitated fatty acids . , . . . .73
The acids soluble in alcohol — kephalophosphoric, kephalic,
and a third acid . . . . . .73
Kephalophosphate of lead . . . . .73
Kephalic and stearic acid, and their barium salts . . 74
Glycerophosphoric acid . . . . .76
Neurin and second oily base . . . . .76
Summary of results of limited chemolysis of kephalin . 76
|3. Complete chemolysis of kephalin by caustic soda . .77
CONTENTS.
xvii
Treatment of the filtrate containing glycerophosphoric acid
and ammonium base
The glycerophosphate of lead .
Acid glycerophosphate of calcium
The platinic chloride precipitate
y. First chemolysis of kephalin with barita hydrate
^ Second chemolysis of kephalin by barita
6. Third chemolysis of kephalin by barita
Z,. Fourth chemolysis of kephalin by barita
r/. Fatty acids produced in the chemolysis of kephalin from
barium salt insoluble in ether
The lead salt insoluble in ether : first, second, and third
preparations
Lead salt soluble in ether
Barium salt soluble in ether, kephalate : product of the first
chemolysis .
9. Product of the secondary chemolysis with barita and caustic
soda in succession ....
K. Theory of the chemical constitution of the kephalins
0. Parami/flin : its isolation, analysis, and compounds
Preparation of free paramyelin from the cadmium chloride
pound ......
Paramyelin cadmium chloride (ox) .
4. Myelin : its isolation, analysis, and cowpounds
General definition of myelin
Modes of obtaining myelin ....
Differences and separation from other cerebral principles
Myelin lead .....
Decomposition of lead salt by hydrothion .
Subgroup of Dinitrogenised Monophosphatides. . N : P = 2 : 1
1. Amidomycdin : its isolation, analysis, and compounds
Process for the isolation of amidomyelin
Amidomyelin dicadm^um chloride compound, insoluble in boiling
benzol from ox buttery after lead process
Preparation of free amidomyelin from the cadmium chloride
pound ....
{a) By the hydrothion process
(b) By dialysis
(c) From the acid mother-liquors
Properties of amidomyelin .
Theory of amidomyelin (ox) as deduced from its cadmium chloride
compound, and comparison with the theory of sphingomyelin
(apomyelin) .......
Diagnosis gind separation of amidomyelin from sphingomyelin
2. AmidokepMdin : its isolcdion, analysis, and comj)Ounds
Amidokephalin- .......
Transformation of this preparation into lead salt .
3. Sphingomyelin, type of the diamidated phosphcUides, ivhich con-
tain no glycerol: its isolcUion, analysis, chemolysis, and com-
IJonnds ........
Process of separating apparently homogeneous crystallised bodies
from the alcohol used for the separation of the cerebrosides ;
which bodies will be shown to be mixtures by reagents .
h
103
104
104
105
105
106
xviii
CONTENTS.
pa<:e
Properties of the product . . . . . .107
Preliminary elementary analyses ..... 107
Comparison with this rosette or lycopodium-like body of a similar
body obtained from the cerebrin mixture by ether, together with
the kephalin, etc. . . . . . - . 107
Chemical and physical properties . . . . .108
Analyses ........ 108
Isolation of sphingomyelin by cadmium chloride process . . 108
Gradual purification of the cadmium chloride salt of sphingomyelin
by recrystallisation from boiling spirit and extraction with boil-
ing ether ........ 109
Further purification of the compound by ether, and by recrystal-
lisation ........ Ill
Elementary analyses of cadmium chloride salt . . . Ill
Preparation of pure sphingomyelin from its cadmium chloride
compound . . . . . . . 112
Removal of the cadmium by diffusion and dialysis . .112
Physical and chemical properties of sphingomyelin . .113
Elementary analyses of sphingomyelin . . . .114
Comparison of sphingomyelin with apomyelin from human brain . 114
Chemolyses of sphingomyelin, with a view of ascertaining its
chemical constitution : experiments 1-4 . , . .115
Theoretical results of these chemolyses . . . .117
Compounds of sphingomyelin . . . . .118
Compounds of sphingom^'^elin with cadmium chloride . .119
C. Subgroup of Dinitrogenised Diphosphatides. N: P = 2 :2 .120
Assurin : its isolation and analysis . . . . ,120
Assurin hydrochlorate platinum chloride . . . .120
Synopsis of the results of the first and second series of analyses and
theory ........ 121
D. Subgroup of Nitrogenised Phosphatide-Sulphatides . . 122
Body from group of cerebrinacides . . . . .122
E. Subgroup of Nonnitrogenised Monophosphatides . . 122
E. Comparative consideration of other Phosphatides of the
Animal Body ....... 12->
Phosphatide of the milk, lactophosphatide, casein . . . 12:J
Phosphatides of the bile, cholophosphatides ; blood, hematophos-
phatides ........ 124
Phosphatides of nucleolar centres of growth (bioiDlasm, cells, etc.),
cytophospliatides . . . . . . . 12r>
(4. Inorganic Bases existing in the Brain in combination with
Phosphatides . . . . . . .126
The precipitate . . . . . . •12''
The ammoniacal filtrate ...... 127
Distribution of the bases and phosphoric acid . . .128
H. Special Study of GtLycerophosphoric Acid and its Salts, as
obtained from some Phosphatides of the Braix . • . 128
Glycerophosphate of lead . . . . . .128
Glycerophosphate of calcium . . . . ,129
Acid glycerophosphate of calcium . . . . .129
Glycerophosphate of barium , . . . .130
The alcuholo-hydrated barium glycerophosphate . . .131
CONTENTS.
XIX
IV.
(iROUP OF NITROGEXISED NONPHOSPHORISED PRINCIPLES.
PACE
A. Subgroup of the Cerebrosides . . . . .134
General properties of the subgroup . . . . .134
1. Separation of the cei-ehroside principles of the braiii , . .136
Spirit treatment . . . . . . .136
Lead acetate treatment . . . . . ,136
Absolute alcohol treatment without fractionation of precipitate . 137
Separation of phrenosin and kerasin by fractional precipitation on
cooling . . . . . . . .137
2. Phrenosin and its derivates . . . . , . 138
a. Further purification of phrenosin by cadmium chloride, ether,
and hydrothion , . . . . ,138
Elementary analysis of phrenosin . . . .139
Consideration of the methods of analysis . . .139
h. Cheraolysis of phrenosin by sulphuric acid in watery solution , 141
Introduction . . . . . . .141
The apparatus. — The leaden tubes. — The hot-air stove . .141
Preliminary purification from inorganic salts of the phrenosin to
be chemolysed . . . . . . .142
Chemolysis of the purified substance . . . .142
The acid filtrates . . . , . . . 142
c. Cerebrose, a new crystallised sugar . . . .143
The crystals. — Cerebrose . . . . . ,143
Reducing power of cerebrose over cupro-potassic tartrate . 143
Polarising power of solution of cerebrose . . ,144
Other properties of cerebrose . . . . .144
The uncrystallised cerebrose . . . . .145
Cerebrosic acid . . . . , . .145
Synopsis of analyses of barium cerebrosate . . .146
Transformation of barium salt of cerebrosic acid into zinc salt . 147
Attempt to produce cerebrosic acid from free cerebrose . .147
Caramel obtained in the chemolysis of phrenosin in which cere-
brosic acid was formed . . . , .147
d. Sphingosin, a new alkaloid, as sulphate, and fatty acids . 149
Removal of the sulphuric acid by caustic alkali . . . .149
Sphingosin sulphate and hydrochlorate .... 150
Consideration of the general chemical function of sphingosin . 151
Neutral, acid, and basic salts ; bearing of the sulphate . .151
Bearing of the hydrochlorate . . . . .152
Sphingosin with potash . . . . . .152
Separation of psychosin from sphingosin . . . .152
Fatty acids and matters soluble in ether, being products of the
chemolysis which yields sphingosin . . . .153
e. Psychosin, its properties and metamorphoses . . .154
Analysis of psychosin . . . . . . 154
Psychosin sulphate . . . . . . 154
Psychosin hydrochlorate . . . . . .155
Psychosin and ammonia . . . . . .155
Chemolysis of psychosin by dilute sulphuric acid . .155
h—'2
XX
CONTENTS,
PAOE
Purple reaction with oil of vitriol . , . .156
Isolation of the purple products . . . . .158
Caramel of psychosin , . . . . .158
Tabular view of the data concerning the caramel of psychosin . 158
Remarks on the caramels . . . . .158
f. Intermediate products of the chemolysis of the cerebrosides with
sulphuric acid : hydrated phrenosin, sesthesin, psychosin . 159
Crystallised soluble in ether product. — .-Esthesin . .160
(J. Chemolysis of phrenosin by sulphuric acid in alcoholic solution ;
formation of ethylic neurostearate . . . .162
The process . . . . . . .162
Neurostearic ether ...... 162
Psychosin sulphate ...... 164
h. Action of heat upon phrenosin ; formation of a caramel . 164
Preliminary experiments . . . . . .164
First experiment . . . . . . .165
Second experiment . . . . . .166
Tabular view of the data concerning caramel of phrenosin . 167
i. Special reactions of phrenosin . . . , .167
Reaction of phrenosin with oil of vitriol, chloroform and glacial
acetic acid ; spectral phenomena of the product . .167
Reaction of phrenosin with Pettenkofer's test ; and spectrum of
its product ....... 167
Ic. Theory of the chemical constitution of phrenosin . .168
3. Kerasin, the second cerebroside : its isolation and properties . 172
Introduction . . . . . . .172
Mode of isolation . . . . . . .172
Mode of purification ...... 173
Special consideration of the properties of kerasin which are made
use of for its isolation . . . . . .174
Reactions of kerasin . . . . . .175
Elementary analyses and theory of kerasin . . .176
Chemolysis of kerasin . . . . . .177
Analysis of the sulphate obtained, and comparison with the com-
position of psychosin sulphate and sphingosin sulphate . .177
B. Subgroup of the Cerebrinacides, or Cerebrin Bodies which
COMBINE WITH METALLIC OxiDES . . . .178
1. General observations on the subr/roup . . . .178
Separation of these substances from the cerebrin mixture by lead
acetate and ammonia . . . . . .178
The lead salts . . . . . . .178
2. Cerebrinic arid : if-s isolfdlon and jjvopertir.s . . . 179
Caramel of cerebrinic acid : first and second experiments . .179
Third and fourth experiments . . . . .180
Tabular view of the data concerning the caramels of cerebrinic acid . 1 80
3. Sulphurised principles, spliero-eerehrin, and others . . 180
Mode of separating these substances from the cerebrin mixture . 180
The crude lead compound (ox-cerebrins) . . . .181
(^uantation of lead and organic matter and of sulphur and phos-
phorus in the dark lead salt . . . . .181
Attempt to isolate the sulphurised principle . . .181
Decomposition of the dark lead salt (ox-cerebrins) by oxalic acid
in boiling spirit ...... 182
CONTENTS.
XXI
Insoluble lead salt (oxalate)
The organic matter from the lead salt
4. Spherocerebrin .....
5, Principal and second product from darh lead salt
The principal product from dark lead salt (ox-cerebrins)
Synopsis of the percentages found and the hypothesis applied
C. SuBrxKoup OF Cerebrosulphatides or non-albuminous Immediate
Principles Containing Sulphur as an Essential Ingredient
Introduction ......
Human mixed cerebrins— barita process applied for their separa
tion
The process .....
Nature of the precipitate produced by barita
Matters soluble in cold benzol
The barita-compound soluble in cold benzol and insoluble in alcohol
Synopsis of data .....
D. Subgroup of Amidolipotides, or Nitrogenised Fats
1. Bregenin :' its isolation and properties
Mode of isolation .....
Physical and chemical properties of bregenin
Synopsis of analyses and theory .
2. Krinosin : its isolation and j)7'operties
Mode of preparing krinosin
Synopsis of the means of elementaiy analyses and theory
formula . . ....
E. Subgroup of Alkaloids . . . .
1. Alkaloids from the human brain
1. Hypoxanthin .....
2. Second alkaloid ....
Synopsis and computation of analyses .
3. Third alkaloid .....
2. Alkcdoids contained in ox brain .
Analyses and theory ....
Computation of analyses ^ . . .
E. Subgroup of Amido- Acids and Imides
1. Leuciu and allied bodies ; tyrosin
Portion soluble in alcohol , . . .
Portion insoluble in alcohol . . .
Note on a peculiar potassium salt
V.
GROUP OF IMMEDIATE PRINCIPLES, COMPOSED OF THREE
ELEMENTS ONLY: ALCOHOLS, CARBO-HYDRATES, AND
ACIDS.
A. Subgroup of Alcohols . . , ... .199
Cholesterin . . . . . . .199
Reaction of cholesterin with oil of vitriol and chloroform ; spectral
phenomena of the product ..... 200
Reaction of cholesterin with oil of vitriol and glacial acetic acid . 201
xxii
CONTENTS,
B. Subgroup of Carbohydrates . . , . , 202
Inosite ........ 202
Compounds of cerebral inosite with cupric oxide . . .202
Compound of inosite from ox brain .... 203
Compound of 'inosite from human brain . . . .201
C. Subgroup of (Nonnitrogenised) Organic Acids . , . 205
1. Lactic acid ....... 205
Summary of analyses of zinc lactate from human brain . . 205
Summary of analyses of zinc lactate from ox brain . . 206
Physical peculiarities of the lactic acid from brain and its zinc salt 206
Peculiarities of the calcium salt of lactic acid from human brain . 208
2. Formic acid . . . . . . .209
.3. Succinic acid . . . . . . . 209
(a) In ox brain ....... 209
[h) In the human brain ...... 210
YI.
GROUP OF ALBUMINOUS PRINCIPLES.
Chemolysis by barita of neuroplastin from brain . . . 212
Nitrogen as ammonia from neuroplastin . . . .215
Total of insoluble barium salts obtained from 10 g. neuroplastin . 215
Barium retained by amido -alkaloid-acid, mixture . , 215
Quantation of acetic acid and tyrosin from neuroplastin . .216
Leucins from 100 g. of ox-neuroplastin . . , . 216
C^uantation of copper in the three products . . . 216
Organic body from mercuric chloride and soda precipitate . 217
Properties of common tasteless leucin and of its copper com-
pound, studied with a view of establishing its diagnosis from
its isomer glycoleucin . . . . . .217
Quantations of solubility . . . . . .217
Stability and regularity of composition of leucin copper compound 218
Leucin obtained by crystallisation from amido-mixture from
chemolysis of neuroplastin . . . . .220
Glycoleucin, the first chemolytic isomer of leucin, its properties
and combinations ...... 221
Solubility of the cupric salt of glycoleucin in cold and. boiling
water ........ 222
Elementary quantation of glycoleucin, prepared from insoluble
copper salt ....... 222
Synopsis of theory and data ..... 22o
Solubility in water at 18° . . . . . . 223
A new reaction and compound of cerebral tyrosin . . 223
Synopsis of data and theory ..... 224
New alkaloids obtained from neuroj^lastin by cKemolysisi . . 225
Mode of isolation of alkaloids from amido-mixture . . 225
Separation of the alkaloidal matter into two groups b}' absolute
alcohol . . . . . . . . 226
Elementary quantation of this new alkaloid . . . 227
Separation of the alkaloidal matter into two groups by cupric
acetate and absolute alcohol . . . . .227
Synopsis of results and theory ..... 227
CONTENTS.
xxiii
YII.
GROUP OF INORGANIC PRINCIPLES, OR MINERAL ACIDS,
BASES, AND SALTS.
Brain Ash , . . . . . . .229
' YIII.
QUANTITATIVE RELATIONS OF THE IMMEDIATE PRINCIPLES
AND CONSTITUENTS OF THE BRAIN.
PAGE
A. QUANTATION OF THE ChEMICAL CONSTITUENTS OP GrEY TiSSUE
FROM THE Human Brain ..... 231
Synopsis of the results of analysis of grey tissue of human brain . 233
B. Quantation OF THE Chemical Constituents of the White
Tissue of the Human Brain ..... 234
Ether extract of the cerebrin deposit .... 234
Synopsis of the results of analysis of white tissue of human brain 236
C. Quantation of the Absolute and Specific Gravities of a
Human Brain and of Several Parts . . . 236
Quantation of the specific gravity of white tissue and grey tissue
of the human brain . . . . . .237
Quantations of the specific gravities of white and grey tissue from
different parts of the brain ..... 238
D. Sketch of a Systematic Quantitative Analysis of the Brain . 242
Analysis of the white matter . . . . . 243
Treatment of the ether solution ; separation of its ingredients
from each other ...... 243
Treatment of the insoluble in boiling spirit part (of the ether
extract boiled with lead acetate) .... 244
Treatment of the soluble in spirit part . . . .244
Separation of cholesterin from myelin lead . . . 244
Solution in spirit of lecithin, cerebrol, yellow colouring matter,
neutral matter, and some cholesterin . . . . 245
Chemolysis of the lecithin, etc., mixture with barita . . 245
Products of the barita chemolysis of the lecithin mixture which
are insoluble in water ...... 245
Treatment of the alcoholic solution which has deposited the white
matter . . . . . . . .246
Treatment of the concentrated alcoholic solution which has depo-
sited the buttery matter . . , . . .246
Analysis of the united buttery and last oily matters . . 247
Chemolysis of the last residue of the buttery and last oily matter
which was not precipitated by lead acetate . . .247
Separation of the ingredients of the buttery matter by a process
in which caustic barita is employed . . . .249
Analysis of the cerebrin mixture ..... 250
E. A Preliminary Experiment for the Quantation of Consti-
tuents OF AN Entire Human Brain .... 252
Quantation of the ingredients of the left hemisphere . . 252
Quantation of the ingredients of the right hemisphere . . 254
Quantation of the ingredients of the cerebellum . . . 255
Quantation of the constituents of the mesenkephalon . . . 255
Table showing results of the quantitative analysis of a human brain 257
A
TREATISE
ON THE
CHEMICAL CONSTITUTION OF THE BRAIN.
I.
INTRODUCTION.
The following work is a contribution towards a system of chemical
statics of the brain, and treats in the first instance of brain matter
as a whole, without separating white from grey rnatter ; it isolates
and scrutinises the several chemical constituents of nerve-matter,
and endeavours, by the study and consideration of the peculiari-
ties of each, and their combination, to obtain sufficient insight
into normal and abnorm-al chemical functions to enable us in time
to guide or to correct them.
It is thus found that this apparently so simple nerve-marrow,
or neuroplasm, is a compound and mixture of a large number of
heterogeneous principles arranged in such a manner as to vanish
completely from appearance as chemical individuals ; the com-
pounds so interpenetrate each other that the resulting material is
aj^parently homogeneous, during life completely so, when seen
with high powers of the microscope, and although in death the
homogeneity partly vanishes, yet even the appearance of the
cylinder axis cannot be utilised chemically at present, and the
isolation and recognition of any ingredients is entirely dependent
upon most circumstantial chemical proceedings.
During these proceedings the first striking fact which meets
the inquirer is that neuroplasm contains abundance of water.
This, in conjunction with the peculiar manner in which the water
1
CHEMICAL CONSTITUTION OF THE BIIAIN
is contained, engenders a mobility of ultimate particles within
certain limits of movement. It also gives penetrability by liquid
diffusion, while excluding porosity and its caj)illary effects ; by
which means a ready nutrition by diffusion in one direction, and
ready cleansing from the effete crystallisable products of life in
another, are ensured. Consequently the brain as a whole is
essentially made up of colloid matter, and may be compared to a
colloid septum, on the one side of which is arterial blood and
cerebrospinal fluid of the ventricles, on the other side, however,
is cerebrospinal fluid of the arachnoideal space and venous blood.
It follows from this that the large amount of water present in the
brain is not there, so to say, mechanically only, like water in a
sponge, and capable of being pressed out mechanically, but is
chemically combined as colloid hydration water, or better, water of
colloidatlon.
All soft organs of the body contain about three quarters of
their weight of colloidation water, fixed within the organised
limits of cells and fibres of all kinds. Cells and fibres are thus
apparently not very different in respect of their mechanical con-
dition from brain matter. Indeed, the difference is less in the
manner of the condition than in the agents by means of which
the condition is brought about. It is in effect mainly by the
number and nature of these agents that brain matter is distin-
guished from other colloid tissues. It contains a considerable
amount of an albuminous l)ase. Whether this is distributed in
the form of sheaths to fibres of nerve marrow, or whether it is
laid into hollow spaces between the fibres, and acts as a cement —
whether it is mixed or combined chemically with the rest of the
matters constituting nerve marrow — must be discussed later. It
may be present in all forms, but does not seem to be present in a
liquid unattached form, as in serum. It appears that the bearing
of soluble albumen when placed in presence of some of the pecu-
liar brain matters changes from that which it ordinarily observes,
no doubt by an influence of these brain matters amounting almost
to combination. The bearing of albumen in the brain being thus
seen to be governed by the matters peculiar to the brain, the
present researches have in the first instance not been directed
upon the condition and nature of the albumen in the brain, but
upon the peculiar matters which seem not only to govern the
albumen, but by their manifold chemical affinities assist promi-
introduction:
3
nently in producing its singular mechanical arrangements, chemical
function, and sensory and volitional action and reaction.
It is not asserted that these matters, or any of them, occur exclu-
sively in brain matter, but being present in the nerves, they
consequently can be extracted from all sensitive or contractile
tissues. They, or some of them, also occur in aggregations of
loose cells, such as the blood-corpuscles and pus-corpuscles ; others
are present in serum, and others again in secretions, such as bile ;
but the quantities in which these bodies are met with in parts
and matters other than those of the nervous system is very far
less than that in which they occur in the nervous system.
The great quantity of these matters occurring in the brain
forms three groups ; the members of one contain at least five,
sometimes six, elements, amongst which , is phosphorus ; hence
they may be termed phosphorised bodies. The members of
the second group contain four elements, amongst them nitrogen,
but no phosphorus, and therefore are termed NITROGENISED
BODIES. The members of the third group contain only three
elements, carbon, hydrogen, and oxigen, present also in the other
two groups, but neither phosphorus nor nitrogen, and ma}^ be
termed oxigenised bodies.
The group of the phosphorised bodies contain the phosphorus
in the form of phosphoric acid combined with from two to five
organic compound radicles. As the. earliest known body of this
group, kcithin, yielded its phosphoric acid mainly in combination
with glycerol, as glycerophosphoric acid, it was supposed that the
phosphorised bodies, of which a number were theoretically
admitted to exist, were constituted like the fats, by combination
of compound organic radicles with the radicle of glycerol, in other
words, that they were ethers of the alcohol glycerol, and con-
tained the phosphoric acid as an inserted, and not as a funda-
mental radicle. But as we now know at least one phosphorised
principle from the brain which does not contain any glycerol, and
does therefore not yield, on chemolysis, any glycerophosphoric
acid, but phosphoric acid merely without any attached organic
radicle, we thereby obtain a new insight into the chemical consti-
tution of the phosphorised substances altogether, and are under
the necessity of subjecting their theory to a revision. According
to the result of this revision the phosphorised substances are not
glycerides at all, as commonly defined, and have nothing in
1—2
4
CHEMICAL CONSTITUTION OF TEE BRAIN.
common with fats considered as glycerides, except that some of
them contain certain fatty acids also present in fats, while they
differ in physical and chemical properties widely from fats. In
accordance with this new knowledge, I have termed the phos-
phorised substances phosphatides, that is to say, substances which
are similar to (but not by any means identical with) phosphates,
on the assumption that their basal or principal joining radicle is
that of phosphoric acid, and that in this acid one, two, or three
molecles of hydroxyl may be replaced by radicles of alcohols,
acids, or bases, and that to a molecle formed by three such sub-
stitutions there may yet be attached, either by substitution of an
element in a radicle itself already substituted (side-chain), or by
addition with elimination of water from the added radicle, a
fourth radicle, and that thus bodies of the following typical
formulse may be produced :
Phosphoric Acid.
j HO (Hydroxyl).
. (Phosphoryl) OP HO (Hydroxyl).
( HO (Hydroxyl).
Nonnitrogenised Phosphatide.
Example : Kephalophosphoric acid.
l' Kephalyl.
(Phosphoryl) OP Stearyl.
I Glyceryl.
Nitrogenised Phosphatide.
Example : Lecithin.
i Oleyl.
(Phosphoryl) OP Margaryl.
I Glyceryl.
Neuryl.
Lin itrogenised Phosph a t id e.
Example : Amldomyelin.
i Acid radicle.
(Phosphoryl) OP Alcohol radicle.
( Alkaloidal or basic radicle (substituted).
I
Alkaloidal anhydride (attached as side-
chain).
INTRODUCTION.
5
The bodies to which the foregoing formulae may be applied
contain the phosphorised radicle once, and may therefore be
termed monophosphatides ; but there are present in the brain and
other protoplastic centres, bodies which contain the phosphorised
radicle twice, and which may therefore be described as diphospha-
tides ; the immediate principle representing this subgroup contains
about seven per cent, of phosphorus, and may perhaps be consti-
tuted according to the following formula :
Dinitrogenised Dipliosphatide.
Example : Assurln.
Acid radicle. ) | Acid radicle.
Alcohol radicle. ■ PO— OP Alcohol radicle.
Alkaloidal radicle. ) ( Alkaloidal radicle.
I shall not in this place dilate upon the nonnitrogenised phos-
phatides, of which I have given a product as an example, although
there are probably two such immediate principles contained in
that part of the spirituous brain extract which is conveniently
termed the buttery matter ; but I shall at once pass to a short
consideration of the mononitrogenised monophosphatides, of
which lecithin is the earliest known, and was, before the institu-
tion of my researches, the only one of which any closer know-
ledge existed.
In this subgroup nitrogen is to phosphorus in the proportion of
one atom to one atom, a relation to be expressed by the formula
N : P 1 : 1 . Of the educts of the brain four species with all their
varieties belong to this subgroup, namely, the lecithins, kephalins,
paramyelins, and myelins ; a product also may be alluded to,
obtained from a dinitrogenised educt by the loss of a nitrogenised
radicle, namely, splilngomyelic add. This latter contains no glycerol ;
the Jecithins, and kephalins, and paramyelins probably contain
glycerol, and yield glycerophosphoric acid; of the myelins, the
constitution in this particular respect has yet to be ascertained.
Lecithin, originally obtained from and named after its occur-
rence in eggs, is only with difficulty evolved from the brain, on
account not only of the many stages of the processes necessary
for its isolation, but also on account of its readiness to decompose
under certain conditions. This tendency is greatest in the
presence of hydrochloric acid and platinic chloride, with which
lecithin readily combines. It ceases almost entirely when lecithin
6
CHEMICAL CONSTITUTION OF THE BBAIN
is combined with cadmium chloride, and the compound is dried.
In the free state and in concentrated solutions, it has an apparently
spontaneous tendency to change and assume colour, due no doubt
to the known tenderness of the oleyl radicle by which it is
characterised ; but the tendency to apparently spontaneous lysis
into proximate nuclei is not so great as is supposed by most
authors, and can now be almost entirely obviated by the improved
processes for its isolation, which I shall have to describe below.
It is, however, the most easily decomposable member of this
group, and this lability furnishes a valuable key to the explana-
tion of many changes in the sick body, which may arise, or have
been proved to arise, from its decomposition. I have not hitherto
found reason to suppose that there is a dinitrogenised lecithin
present in the brain ; but in view of the dinitrogenised educts
which will be described, the possibility of this occurrence must
not be lost sight of.
I have given the explicit formulae of the lecithins considered
as phosphatides under the chapter relating to them ; the con-
tracted formulae are :
Oleo-i^almito-glycero-neuro-phosphatide = 0^.2^82^^^)^.
Oleo-margaro-glycero-neuro-phosphatide = C43Hg4NPO^.
Oleo-stearo-glycero-neuro-phosphatide = C^^Hg,^.NPOg.
Of the second lecithin, there have been analysed the following
salts :
Cadmium chloride salt
Hydrochlorate - . -
Hydrochlorate platino chloride
- C,3H,,NP0,-{-CdCl,.
- 2(C,,H,,NP0, + HCl) + PtCl,.
In the chemolyses the oleic, margaric, palmitic, and stearic acids
were isolated, oleic acid characterising the phosphatide ; stearic
acid was always present in very small quantities only ; it was
probal.)le that of stearic acid the two isomers discovered in these
researches may sometimes be present in lecithins. Regarding the
l eputed identity of margaric and palmitic acid, I still entertain
some doubts, which have been rather increased than diminished
by the discovery of the isomers alluded to. Besides these acids
the lecithins always yielded glycerophosphoric acid and neiirin.
A specimen obtained from cadmium chloride salt soluble in cold
INTRODUCTION.
benzol, was recently analysed and found to answer to the fore-
going description.
As the lecithin species is characterised by the presence of the
radicle of oleic acid, so the kephalin species is characterised by
the presence of the radicle of a peculiar acid, to which I have
given the name of kephalic acid. This acid is even more changeable
than oleic, and imparts its quality to all the compounds in which
it is present. The change in these cases seems to be either
acquisitive, e.g. by accession of oxigen, or intramolecular, e.g. by
transposition of atoms, but does not appear to lead to lysis into
proximate nuclei so easily as in the case of lecithin. The second
fatty acid radicle in the case of the principal kephalin is stearyl,
other radicles occurring only in extremely small quantities. The
members of this subgroup vary in the amount of oxigen which
they exhibit on analysis, in a manner so as to be apparently sharply
characterised thereby. But this variability of the constituent
oxigen may be transitional, and, on the whole, this remarkable
feature, which none of the other phosphatides exhibit, requires
much further investigation before it is adducible to any very
precise theory.
A kephalin mav therefore be defined as kephalo-stearo-glycero-
neuro-phosphatide, and represented by the formula
To be a kephalin a phosphatide must contain the radicle
kephalyl, or a homologue, which governs most of the properties of
the compound ; its peculiar properties prevail over those of the
second acid.
A kephalin with palmityl, Cj^Hg^O^, in place of stearyl, would
have the summary formula, C^^H^^NPOg + aq. ; a kephalin with
margaryl, CjwHggO^ would be C^^H-oNPOc, + aq. If there were
several homologous kephalic asids, such as some analyses seem to
indicate, then for a kephalyl of formula C^wUgQC.:,, combined with
either stearyl, margaryl, or palmityl, the foregoing formulae would
have to be increased by CH^ each, so that the most complicated
kephalin might contain 44 atoms of carbon. None of these hypotheses
explain either the deficiency of hydrogen or the excess of oxigen
/ Ci,H,A (kephalyl) )
OP
■-C,3H3,NPO, + aq.
8
CHEMICAL CONSTITUTION OF THE BRAIN.
(namely, as compared to the theory derived from chemolysis) in the
various kephalins and their compounds which have been analysed,
and of which the following is a synopsis :
Kephalin (empirical formula) - C^2^79^^^i:r
Kephalin, perhaps - - - C42HgJ»NPOg + 5H.p.
Kephalin cadmium chloride - C42H^9NPOj3 + CdCl^.
(dissociates partially in watery reagents.)
^''^Sm Srid'e"™'! } 2(C,,H,,NP0„ + HCl) + Pt CI,.
Kephalin with amido-kephalin | C^2Hg,^N.^POj3.
(mixture) - - - j 2(C^2H79NPOi3.
Oxykephalin cadmium chloride C42HK9NPOJ4 + CdCl^.
Peroxykephalin - - - C42H;9NPOif^.
Peroxykephalin, diplumbic - C^g^^^s^^^^P^r/
Kephaloidin - - - - C^^H^^NPOig.
^""^^chbrtde^^^ cadmium J 2(C^2Hr5NPO,,) + CdCl2.
There is little doubt of the existence of a dinitrogenised
kephalin, termed amidokeplialin, which was mixed to the extent
of one-third nearly with a preparation of kephahn met with in
the course of the researches. This body is less soluble in ether
than kephalin. It is probably constituted analogously to the
amidomyelin described in a special chapter. But as this amido-
kephalin has not been finally examined, I am unable to give any
further data concerning it.
Of the decomposition products of kephalin by chemolysis, I
have already mentioned Irj^halojihosphoric acid, under the non-
nitrogenised monophosphatides. Kephalk acid is the principal
chemolytic product of the process, in w^hich the phosphatide is
almost entirely severed. All its salts are soluble in ether and in-
soluble or little soluble in alcohol. It assumes, in the free state
or in combination, a brown colour, from which it has not yet been
possible to free it by any known process. The formulae which
can be constructed for the acid vary between Cj()H3203, and
CJYH28O3, or CjwH3y03, and CJ-H32O3. They are mainly derived
from barium scdts, which contain from 18-28 per cent, to 20*05 per
cent. Ba. in some cases 19 "29 per cent, to 19-89 per cent. Ba.
The kephalates of barium, of calcium, and of lead, are brown
precipitates.
Another chemolytic product of kephalin is stearic acid, of
INTRODUCTION.
9
melting-point 69*5 ; this was examined in the free state, and as
l)arium and lead salt.
Stearic acid - - -
Barium stearate - - '2{(^^^.^.fi.^'Edi.
Lead stearate - - 2{Q^^^fi^)Vh.
Grlycerophosplioric acid was isolated from kephalin as lead salt,
calcium salt, CgH^CaPO^j, and in the new form of add calcium salt,
C^^Hi^^CaP20i2 ; as barium salt, CgH^BaPOg ; this could be
obtained crystallised, monohydrated, and as alcoholohydrate, in
which latter state one molecle of acid glycerophosphate of barium,
composed similarly to the acid calcium salt, contained at least
three molecles of alcohol and six of water.
Kephalin further yielded neurin and another base, perhaps
derived from the former. The second base in amidokephalin was
not ascertained.
The third subdivision of this subgroup is represented by
paramyelin, a phosphatide which contains probably glyceryl,
neuryl, and an oleo-cholide radicle hitherto unknown. It strikes
with oil of vitriol and sugar syrup an immediate deep purple
colour ; the mixture of acids obtained by chemolysis with barita
does the same in a more intense manner. Paramyelin is a white,
firm, solid body, crystallising from boiling spirit in plates and
needles. It combines with cadmium chloride, and the solubility of
this compound in hot benzol, and its insolubility in cold benzol,
afford facilities for the isolation of the body. A specimen of
cadmium chloride salt from human brain gave on preliminary
analysis Cg^H^gNPOg + CdCl^; another from oxbrain,
CggH^gNPOg + CdCl2. It is not maintained that these com-
pounds were unitary and did not contain an admixture of a small
quantity of one or more principles similar to paramyelin, i.e.,
paramyelins differing from each other in the item of the second
acid radicle. Paramyelin also forms a hydrochl orate platino-
chloride salt.
While lecithin and paramyelin exhibit mainly alkaloidal
functions, and kephalin shows alkaloidal and acid functions on a
wide area, the fourth subdivision or species of the mononitrogen-
ised monophosphatides, the myelins, exhibit mainly acid properties.
The representative myelin is a firm compound, and combines with
lead like a dibasic acid ; that is to say, admits a didynamic atom
of lead in place of two atoms of hydrogen. On the other hand, it
10 CHEMICAL CONSTITUTION OF THE BRAIN
does not combine with cadmium chloride as do lecithin, kephalin,
paramyelin, amidomyelin, and s25hingomyelin. This peculiarity
seems to indicate a peculiar constitution, of which, without ex-
haustive chemolyses, no account can be given, but which may be
such as to necessitate the separation of myelin from the other
three members of the subgroup, and its allocation to a subgroup,
of which it would be the peculiar representative. Myelin and its
compounds on analysis have jdelded the following formulae :
Myelin in lead salt - - - C^^H^-NPOk).
Myelin free - - - . Cgc^HlNPOg.
Myelin lead . - . . C.I.H^gPbNJPOiy.
There may, perhaps, be myelins varying in carbon from 39 to 44
atoms.
I now come to the subgroup of dinitrogenised monopJiosphatldes, of
which I had given a preliminary notice in my researches of 1874
under the name of amidomyelin and apoDiijelin, but of which the
former has been isolated only latel}^, while the latter has been
more fully investigated, and classified with a body isolated and
investigated under the name of sphingomyelin. Amidomyelin
completely bore out the theory of the composition which I had
assigned to certain compounds of mixtures of brain-educts with
platinum and cadmium chloride ; out of such mixtures amido-
myelin on the one, and lecithin and paramyehn on the other hand,
were isolated first as compounds, afterwards in the free state. If
any argument were needed to justify the analyses of the mixtures
alluded to, analyses to which no fairminded inquirer would attribute
any other character than that of reconnoitring proceedings, the
success which has been induced by their results would be sufti-
cient. Apomyelin was found to be a genuine educt and imme-
diate principle of the brain.
Of amidomyelin I have, therefore, practically j^roved the existence
and individuality by direct isolation and analysis. But I have
not had time to ascertain anything about its constitution by
chemolysis of the pure substance. There are, however, some data
available for a preliminary view of at least some of the radicles
likely to be met with in the l)ody, in a research on the chemolytic
products of a cadmium precipitate, which in the relative research
I have described as the principal cadmium salt.
Amidomyelin is analogous to sphingomyelin and apomyelin in
this, that it contains two atoms of nitrogen upon one atom of
INTRODUCTION.
n
phosphorus ; these two atoms of nitrogen are disposed in two
different radicles, and influence the character of the compound so
that it presents itself as a diacidic base, or a dipolar alkaloid. In
the latter quality it combines with cadmium chloride in two ratios,
the fully saturated compound, which is that most commonly ob-
tained, containing a little more than 30percent. of cadmium chloride.
Amidomyelin has been proved to exist in five forms, one of
isolation, four of combination.
Amidomyelin - - - - - C^^HggN^PO,,.
Amidomyelin hydrochlorate - - C44HggN2P09 + HCl.
Amidomyelin monocaduiium chloride - C^^HggNgPOf, + CdClg.
Amidomyelin dicadmium chloride - C^^HggN.^POc, + 2(CdCl2).
Amidomyelin hydrochlorate platinum ^ 2(C44HggN2P09 + HCl)
chloride - . . . - \ + PtCl^.
The second subgroup of the diamidated monophosphatidcs in-
cludes sphingomyelin and ajjomyelin. Sphingomyelin has been
much studied and chemolysed, and has given not Only the funda-
mental information upon which the hypothesis of the phosphatides
is based, but also the first knowledge of a number of compound
radicles new to science. It is not maintained that the knowledge
of the bodies, acids, alkaloids and alcohols, in the shape of which
they appear after chemolysis of the principle, is definitely roui^ed
off. For in its prosecution, conditions of the utmost difficulty and
complexity were met with, arising mainly from homology and
isomerism. Thus one product was a fatty acid of the composition
expressed by the formula CigHg^jO^, being the third isomer of stearic
acid discovered in my researches, sphingostearic acid, fusing at 57°,
therefore at almost the same interval below, as the second isomer
of stearic acid, namely, neurostearic acid, fusing-point 84°, fuses
above the fusing-point of ordinary stearic acid, which latter melts
at 69*5'^. It is evident from this that henceforth a fatty acid
from the brain can be diagnosed neither by its elementary com-
position alone, nor by its melting-point alone, but that a know-
ledge of both is required to approximately fix its nature ; an
accurate diagnosis requires the knowledge of all physical and
chemical properties and of reactions. More particularly a mere
melting-point determination of a sample of fatty acid from the
brain has, by itself, no diagnostic value whatever, and particularly
can no longer be used in the attempt to make out, by the well-
known tables of Heintz, the quantities of different ingredients in
12 CHEMICAL CONSTITUTION OF THE BRAIN.
mixtures of which certain melting-points are supposed to be
accurate exponents.
How wonderfully the phenomenon of isomerism complicates
brain research and biological research in general will become still
more apparent by the following. Another product of the
chemolysis of sphingomyelin is an alcohol, sphingoid of the
empirical formula Ci^HjgO, or C^gHg^^O^, which, on the supposition
that the latter formula expressed its atomic weight, would be the
fourth isomer of stearic acid.
A third product of the chemolysis of sphingomyelin is an
alkaloid closely resembling the sphingosin of the cerebrosides,
Cj^Hg^NOo, but showing a little more carbon and hj^drogen, so as
to answer to the formula CqH^^NO^. To these three radicles, a
molecle of neuryl, C^H^^O, is yet attached, so that on the basis
of its chemolysis we are able to attribute to sphingomyelin three
somewhat different formula? of constitution, in each of which three
terms are certain, while of the remaining two terms one is certain
as regards its percentic composition, but uncertain as regards its
atomic Aveight ; while the other, namely the second nitrogenised
radicle, is not yet sufficiently well defined to afford much aid to
synthetical calculations. The smallest formula gives a total of
CgoHjQ^N^PO^, while the largest leads to a formula with 61 Carbon;
but all empirical formulae for sphingomyelin, which we shall have
to consider below, lead to the necessity of assuming the presence
in the free body of a few atoms of water, which are not accounted
for by the sum of the products of the chemical cleavage.
It is certain that the smallest formula just given, and which is
almost identical with the formula for apomyelin formerly given, is
only a type, and that there are varieties in which either the
alkaloid or the fatty acid, or the alcohol differ from those just
formulated probably by — hCH^, or — hnCH^. The increase in
CH^ of the alkaloid obtained by chemolysis beyond the quantities
demanded by the formula of sphingosin may be due to the admix-
ture of a small quantity of a body being a compound of sphingol,
CigHg^O,, and sphingosin, Cj^H.-NO^, ; total, C3^H6c,N03 + H.p.
If all these data are constant, then we cannot doubt that the
limits of error are confined between very small dimensions.
The typical sphingomyelin crystallises well in microscopical
plates, combines with cadmium chloride in two ratios, the com-
pounds being crystalline, and gives up one nitrogenised radicle
INTBODUCTION.
13
on limited chemolysis, namely the loosely bound neurin, C-H^gNO,
leaving an acid which contains all the phosphorus of the sphin--
gomyelin together with half the original nitrogen, and in which,
therefore, N : P=l : 1. The formula of the acid, a produced
mononitrogenised monophosphatide, from the C53 sphingomyelin
is C4gN95NPOj2, and its name may be sphlngomyelic acid.
The following is a synopsis of the bodies belonging to this
subgroup and their compounds and chemolytic products :
Eduds.
Sphingomyelin (ox) - - - . C52Hio4]N'2POc) + H.p.
Apomyelin (man) - - - - C^^H^QgNgPOg.
Sphingomyelin (theory from chemolysis) C^gHj^gNgPO^ + 2H2O.
Sphingomyelin cadmium chloride - C5jHc)()N2PO^Q-f-CdCl2.
Do. dicadmium chloride - C5iHj,9N2POio + 2(CdCl2).
JDerivates by Chemolysis.
Sphingomyelic acid - - - - C4gH95NPOi2.
Sphingosin ----- CJWH35NO2.
Base (as sulphate) - - - - 2(C2o'H4iN02) + H2S0^.
Sphingol ------ CigH3g02 or CgH^gO.
Sphingostearic acid (m.p. 57°) - - C^gH^gO.,.
Neurin ------ CgH^^NO.
Nitrogenised product - - - C.^^HggNO,,.
Phosphoric acid - - - - H3PO4.
The diamidated phosphatides yield hydrochlorates which crys-
tallise from anhydrous solvents in the presence of excess of acid in
exquisite form and purity ; but they are not stable in the pre-
sence of watery reagents, and yield hydrochloric acid to solvents
at every recrystallisation, so as to become almost free from the
acid by mere repetition of this process. When the hydrochlorates
are produced from the cadmium salts, they are of course mixed
with double the quantity of hydrochloric acid necessary for
neutral salts ; and at this point great care is required so to
manage the necessary warming of the mixture, that the salt may
be formed without any very great part of it being chemol3^sed
under the influence of the redundant hydrochloric acid.
The diphosphatide to which I have already alluded, in which
N : P = 2 : 2, and to which I have given the name of assurin (from
the Assyrian God), has in its platinum chloride compound the
formula C4gH94N2P209. It is at present the most phosphorised
body of this class known, while one of the least i^hosphorised,
14 C HE MIC J L CONSTITUTION OF THE BRAIN.
relatively to the nitrogen, is a phosphatide educed from the bile
.as a crystallised platinum chloride ealt in which N : P = 4 : 1,
and which has a formula by which it is characterised as a tetra-
polar alkaloid, the four basic poles corresponding to the four
nitrogenised radicles with which the body is endowed.
Thus I have shown that the phosphorised educts of the brain
are a class of bodies with numerous genera, each genus having
again species and varieties ; that they are of greatly varying chemi-
cal construction and function, and that the}^ are so distributed as
to show that the}^ are specifically concerned in the most intimate
parts and processes of protoplasmic life.
The presence of water diminishes the number or avidity of
affinities in all phosphatides ; it combines itself with these bodies
in a peculiar manner, by which they show their character as
colloids, and it afterwards dissolves them, some in a perfect, others
in a peculiar and imperfect manner. When this hydric colloida-
tion is at a maximum, the tendencies to decomposition seem to be
at a minimum. The watery solutions do not decompose in
stoppered bottles for many months ; only after six months some
specimens so kept showed signs of decomposition or putrescence.
It seems therefore that water satisfies some of those affinities, and
what is most remarkable, its influence increases and diminishes
with the mass which is present and capable of acting, so that it
displaces, when in quantity, other combinants, but when these
other combinants prevail, water is itself displaced, and the colloid
state instantly disappears. In the dilute watery solutions of the
phosphorised bodies therefore almost every reagent soluble in
water, when added in a certain excess, produces a precipitate,
which contains the reagent in combination. But when water, or
any watery solvent capable of dissolving the combined reagent, is
brought in contact with the compound, the compound imme-
diately dissociates ; the reagent passes into the water, jort.!)'^//.
as the phosphorised body passes into the hydrated colloid state,
and if the influence of the water is continued by renewal, the pi-o-
cess terminates by a complete separation ; the phosphorised body
is again free and pure, and swells and dissolves as at first.
The reagents with which the phosphorised bodies are thus able
to combine, and from which they are dissociated by water, are
acids, alkalies, and salts. The phosphorised bodies therefore
possess alkaline affinities (for acids), acid affinities (for alkalies),
INTRODUCTION.
15
alkaloid affinities (for salts) ; all those affinities are overcome by
water in quantity, but the affinities for water are overcome by
some metallic oxides, such as of lead, copper, manganese, iron,
and even to a slight extent by lime and potash ; these latter
compounds are dissociated only by strong mineral acids, and the
compounds can then be dialysed out. All other combinants
separated by water alone can be completely removed from the
phosphorised substances by dialysis on vegetable parchment.
We have therefore here a diversity of affinities such as is not
possessed by any other class of chemical compounds in nature at
present known ; and the exercise of these affinities being greatl}^
influenced by the mass of reagent, and the mass of water which
may be present, the interchange of affinities may produce a per-
fectly incalculable number of states of the phosphorised and
consequently of brain-mattei'. This power of answering to any
qualitative and quantitative chemical influence by reciprocal
quality or quantity we may term the state of labile equilibrium ; it
foreshadows on the chemical side the remarkable properties which
neuroplasm exhibits in regard of its vital functions.
From this it also follows that neuroplasm (if only as charac-
terised by the phosphorised bodies) must yield obedience to every,
even the slightest external chemical influence, which may reach it
by way of the blood. It must take up metals, acids, salts, alka-
lies, and alkaloids presented by the blood ; it can retain only
oxides when the serum is again free from the combinants ; a
watery serum will wash the brain, a more watery one will make
it swell and displace mechanically within physiological limits
what it can ; a still more watery one will make the brain dropsical
and produce some of the conditions of mechanical j^ressure on the
brain. All these processes are the necessary consequences of the
affinities of the phosphorised substances, and these being known,
the phenomena could be predicted, if they were not sufficiently
known as phenomena, though hitherto destitute of an explanation.
Thus the so-called brain-fungus, the continued protrusion of brain-
matter through apertures of the skull produced by mechanical
injuries, may in certain cases find a physical explanation in
simple excessive hydration of the phosphorised (and nitrogenised)
principles, producing general intra- cranial pressure.
These few examples show that the acquisition of chemical
statics leads almost necessarily and very easily to chemical d^aia-
16 CHEMICAL CONSTITUTION OF THE BRAIN.
iTfiics of the brain ; and these will in their turn furnish data for
physiological and pathological conclusions. But these deducive
arguments must be most sparingly and cautiously used, until the
statics are in a state of perfection and completeness. To argue
too far from incomplete data would, seeing the history of biolo-
gical chemistry during the last thirty years, be a deplorable error.
The NITROGENISED NONPHOSPHORISED substances of the brain
imitate in many respects, but with little intensity, the properties of
the phosphorised. Thus some have the affinity for water to the
extent of swelling to gelatinous masses, but they do not go to the
state of apparent solution, and do not pass paper filters under any
l^ressure. Some are insoluble in ether, cold benzol, cold alcohol,
some soluble ; all dissolve in hot benzol or alcohol, and are depo-
sited on cooling almost entirely. Therefore as compared to the
phosphorised bodies, their chemical character is slight solubility ;
from their hydrated colloid state they are also reduced to a more
compact and combined one by many acids, alkalies, and salts, and
they retain many oxides in combination. Water has the same
dissociating influence upon these as upon the compounds of the
phosphorised principles. The nitrogenised bodies are all firm
compounds, and do not easily oxidise or decompose. Their
atoms show very little tension ; but they possess substitution
poles, where hydrogen is replaceable by metalloids, or com2:>ound
radicles ; their compounds with salts, oxides, or acids, are so un-
stable as not to admit at present of quantitative definition.
This group includes six great subgroups, of which the first
four have a great number of features in common, while the two
last subgroups are dissimilar to the former, and much more
simple as regards chemical constitution.
The first subgroup is that of the cerebrosides, or bodies which
contain a peculiar sugar, cerehrose, in which different radicles of
acids and alkaloids (it is not known whether of alcohols also, in
some cases) are inserted. Thus, of phrenos'ut, C^^H^gNOg, the
definition and formula of constitution are the following :
Nenrostearo-Si)Mngoso-Cerehro8ide.
INTRODUCTION.
17
The derivates by chemolysis and synthesis of products are the
following : — Cerebrose, a crystallised sugar, isomer of glucose, and
like this dextrorotatory, reducing copper solution, and tasting
sweet,- of formula C^3Hj206 ^ Neurodearic acid, an isomer of stearic
acid, fusing at 84", of formula CjgH3,302 ; Neurostearic ether,
C^qH^qO^, or {C.flrJ)C-^^'H.ofi.2^ produced during chemolysis of
phrenosin in alcohol by sulphuric acid, can be distilled unchanged
in vacuo; Sphingosm, an alkaloid, C^-Hg^NO^ ; as sulphaie,
2(Cj-H35N02)H2SO^, insoluble in absolute alcohol ; or hydro-
chlorate, Cj-Hg^NO.^HCl, soluble in water ; Psychosm, C^gH^^NO^
being the cerebroside of sphingosin, a body having alkaloidal
properties, but less pronounced than those of sphingosin ; .^Esthe-
sin, a compound of sphingosin and neurostearic acid less water,
Cg.H^gNOg. Under the influence of heat phrenosin yields a
caramel, C^^Hk-^NO^, by the loss of four molecies of water ; by a
similar reaction psychosm also yields a caramel of the formula
C03H.3K.NO.3 ; these caramels are brown, insoluble in spirit, soluble
in ether.
Synopsis of cerebrosides and derivates :
Phrenosin (educt)- - - - C^^H^j^NOg.
Derivate by dehydration; caramel - C^jH-^NO^.
Derivate by substitution : Nitrited / C^iH^gN^Ojg, or
phrenosin nitrate - " - - ( C4iH^g(N02)NOg + HN03.
Derivates by cleavage {chemolysis) and synthesis of products :
Cerebrose ----- C^Hj^O^^.
IN eurostearic acid- - - - C^gHgcO.,.
Ditto ether ' - - - - O.^^Yi^fi.^ or {^"^■2^r;)^i^'^;fP->;
Sphingosin - - _ _ C\-H35N02.
Ditto sulphate - - - - ^(Cj.R.^NO.,) + H2S0^.
Ditto hj/drochlorate - - - Ci^Hg^NO^-f HCl.
Psychosin ----- C.gH.^NO:.
Caramel of psychosin - - - C.23H3^N03.
The second cerebroside, and accompanying phrenosin, is
kerasin, a body crystallising in microscopic filamentous masses,
which are very voluminous, and enclose mechanically great
volumes of spirit. Although the knowledge of this body has
been greatly advanced lately by the discovery of processes for its
separation from sphingomyelin, and more particularly from a body
which I shall describe later under the name of binosin, yet cir-
cumstances did not allow me to finally fix hy chemolysis its
rational constitution in the same manner as this has been done
2
18 CHEMICAL CO.NSTITUTION OF THE BRAIN.
for phrenosin. Kerasin may, like phrenosin, comprise a number
of analogously constituted bodies, of which the most probable
formulae are :
Psychosin (as sulphate) - - 2(C,.3H^.^NO-) + H.^SO^.
Cerebrose ----- CgH^o^G-
Fatty acids of the formula - - CnH^nO.,.
Both kerasin and phrenosin are neutral bodies, and do not com-
bine with acids, alkalies, and salts in stoichiometric proportion.
The cerehrinacides, or cerebrin bodies which combine with lead
and other bases, are as yet little known. They will yet require
long and important researches^ not only because they are numerous,
but more particularly because they occur mixed with a class of
sulphurised bodies, of which I give a preliminary sketch below. It
is probable that the first cerebrinacide to be considered — namely,
cerehrinic acid — ma}^ be a cerebroside, in which three hydroxy] s are
replaced (in phrenosin and kerasin only two hydroxy Is are thus
replaced), two by fatty acid radicles, one by an alkaloid radicle.
The fact that cerebrinic acid became dehydrated like phrenosin
under the influence of heat, assumed a brown colour, lost its solu-
bility in spirit, and acquired a new solubility in ether — in short,
that it became changed in the same manner as a saccharide is
changed when it passes into a caramel — supports this surmise.
Whether others of the cerehrinacides are cerebrosides can at pre-
sent be neither asserted nor denied. Several of the cerehrinacides
which have been isolated, such as spherocerebrin and the (accord
ing to quantity) principal cerebrinacide, are distinguished from
the cerebrosides and cerebrinic acid by their containing a much
larger proportion of oxygen than these bodies. The following is
a synopsis of these bodies as far as they are isolated, together with
their preliminary emj^irical formulae :
Cerebrinic acid - - . . C3,)H^j..N0.,.
Caramel of cerebrinic acid - - Cr,„H^or.^Q3-
Spherocerebrin . . - - C.j.Hjo.,NOj-.
Cerebrinacide, principal - - C^rjHjjgNO^j.
There are a number of subordinate cerehrinacides waiting for
closer investigation.
Educfs :
Kerasin
Ditto
Ditto
Prod acts h)i cJienioIj/sis :
INTRODUCTION.
19
Of the cerebrosulphatides I have hardly done more than demon-
strated the existence. No representative body has been isolated
to the extent of being free from phosphorus and cerebrinacide ;
indeed, it must remain a question whether there are not bodies
which contain sulphur and phosphorus at the same time. The
most concentrated preparation of cerebrosulphatide which I have
succeeded in producing contained 4 per cent, of sulphur.
We now progress to the consideration of an entirely new series
of. bodies, which are so constituted, and exhibit such properties,
that they may perhaps be described as nitrogenisecl fats or amiclo-
lipotides. Although they occur mixed with the cerebrosides, never-
theless they are at once demonstrated not to be such by the low
amount of oxygen which they contain. Of these, the first is
hregenin (from the Low German ' bregen,' head or brain), which is
easily soluble in cold ether, crystallizes, and has the formula
C^oHg^NO.. The second one is krlnosin (from the Greek word for
hair, the wavy crystals resembling a mass of tangled long hair),
CggH-^NOg, insoluble in cold, easily soluble in boiling ether.
Homology of these two bodies, which is suggested by a comparison
of the formulae —
may not be assumed, as the one with the higher number of carbon
atoms fuses at a much lower temperature than the one with the
lesser number of carbon atoms.
Of the group of alkaloids occurring in the brain, hypoxanthin and
its compounds were known to science, but isolated with greater
precision from brain, particularly by the aid of phosphomolybdic
acid. The following bodies were produced and analysed :
Hypoxanthin - - . - C^H^N^O.
Ditto hydrochlorate - - - C5 H^N^O + HCl + H2O.
Ditto with platinum chloride - '2{C^H^^fi 4- HCl) + PtOl^.
Ditto nitrate - - - - C5H4N4O + HNO.,.
Ditto with silver nitrate - - C^H^N^O -f AgN63.
Ditto with silver - - - CgH^N^O + Ag^.
The other alkaloids contained in the brain are either much more
complicated or more simple than hypoxanthin. I have distin-
guished these, pending further inquiry, the second alkaloid as
gladioUn, and the third alkaloid as tennysin ; and regarding them
little more can be stated than the methods by which they were
Bregenin -
Krinosin -
2—2
20 CHEMICAL CONSTITUTION OF THE BBAIN.
isolated, and the elements of which they seem, empirically, to be
composed :
Gladiolin (second alkaloid) ^i^H^QNgOg.
Ditto hydrochlorate 2:old ) n xr at ■ -rrrn ■ a r^^
chloride - - _|CiAoNA + HCl + AuCl3.
Tennysin (third alkaloid) C.3H5NO.
The amidoacids and amides, represented by leycin and allied lyr'in-
riples, and tyrosin, bodies otherwise well known in science, were
isolated from healthy brain for the first time in the course of
these researches :
Leucin - - - - C,5Hi.,N02.
Tyrosin - - - - CyHiiNOg.
Urea was isolated from brain and cerebrospinal fluid, particu-
larly in disease. In cases of cholera I found that the cerebrospinal
fluid contained as much urea as healthy human urine — namely,
about 2 per cent.
The group of oxigenised nonnitrogenised pinc'iples consists mainly
of alcohols and organic acids. The alcohols have very slight com-
bining powers. The most prominent of these, as regards both
quantity and appearance, is cholesterin. Discovered originally in
human gallstones, and erroneously believed to be a fat, this prin-
ciple received the inappropriate name which hides its significance
and character. Insoluble by itself in water, it is probably dis-
solved in neuroplasm by means of the phosphorised substances.
Its bearing is therefore governed, and its role determined to a
great extent, by these matters. But its intrinsic chemical dynamis
is probably also independent to some extent, as its atomic weight
is very high. As an alcohol it is monodynamic, and if it did
combine with acid radicles, naturally would therefore give rise to
one class of ethers only. If such compounds exist in the brain,
they must be in a state of high atomic tension, and fall to pieces
by the mere fact of the application of solvents.
Some data seem to point to the existence of a second alcohol
by the side of cholesterin, and isomeric or homologous to it, but
the hypothesis lacks definite proof. I have sometimes found
cholesterin fusing at 137°, the point at which some earlier writers
supposed cholesterin always to fuse. But since the fusing-point
of the principal cholesterin is certainly 145^, the lower fusing-j^oint
of ViT may belong to an isomer or homologue, and this I propose
to term jjhrenoster in. . .
INTBODUCTION,
21
The bodies which may conveniently be considered as forming the
subgroup of carhohydrates are inosite, the isomer of dextroglucose,
discovered in and bearing its name from muscle, and glijcogm,
discovered in the liver and other organs. It has seemed to
me that the inosite from human brain was somewhat different
from that of the ox; at least, while the latter easily gave a com-
pound with cupric oxide of the formula C^^H^.^O^ + 3CuO + SH^O,
the former gave no such precise combination, and reacted in a
peculiar manner. Some have supposed inosite to be the material
from which, by a post-mortem metabolism, the lactic acid of the
bjain took its origin. But since it has been shown by others that
inosite by ferments yields ordinary zymolactic acid, whereas, as I
have shown, the lactic acid of the brain is always and exclusively
the optically active paralactic or sarkolactic acid originally dis-
covered in flesh, this apprehension has become obsolete.
The names and formulae of the bodies belonging to this sub-
group are the following :
Cholesterin, m.p, 145° -
Phrenosterin, m.p. 137° -
Inosite - - - -
Ditto tricupric trihydrate
CgHi^O^ + 3CuO + 3H2O.
The subgroup of the nonnitrogenlsed organic adds is represented
by at least four different principles — formic^ sarkolactic, succinic,
and a tribasic acid which may perhaps be termed oxyglyceric.
Formic acid is present in such small quantity only that it can just
be recognised in the distillate by reactions. But sarkolactic acid
occurs in quantities up to 1 per mille of the fresh brain substance.
It was thus possible to subject this body to a more intimate study,
and describe its properties with great precision. Succiiiic acid was
separated and identified in a satisfactory manner. The fourth
acid, oxyglyceric, was isolated and studied mainly by the aid of its
silver salt. The following is a synopsis of these acids, their for-
mulae, and some of their compounds :
Formic acid _ _ . _ CH.fl.^.
Sarkolactic acid - ' - - CgH^^O^
Ditto zinc salt, hydrate - - Zn(C.3H503)^-}-(H20),.
Ditto calcium salt, anhydrous - CaiCgH^Ogig.
Succinic acid - - - - C^HgO^.
Oxyglyceric acid - - - CgHgO^.
Ditto silver salt, tribasic - - CgH^Ag^O^.
22 CHEMICAL CONSTITUTION OF TEE BRAIN.
The alhminous siibstances of the brain may be considered as
nitrogenised sulphatides, inasmuch as sulphur is an essential con-
stituent ; if bodies like casein were present in brain, as has been
supposed by some inquirers, they might, like the casein of milk,
be true nitrogenised sidphatide-phospliatides ; if connective tissue
were' present, it would perhaps come under neither definition.
These substances are as yet very little known, for they could be
examined with advantage only after all the substances described
in the foregoing pages were known and could be separated. But
in this process the albuminous bodies undergo such important
alterations that the difficulty of their study is thereby only altered
in kind, but not diminished in amount. With due consideration
to this circumstance, they were in the first instance studied by
chemolysis, and have yielded very remarkable results. The deri-
vates by chemolysis included the following :
Albuminol CigH^^NO,. (?)
Volatile sulphurised body
Ammonia and compound am- ( -^rjj
monias ----- j s-
Carbonic acid - - - - CO^.
Fatt}^ acid - - - - . - — —
Oxalic acid ----- C.^H^O^.
Phosphoric acid - - - - H.^PO^.
Sulphurous acid - - - - SO.^.
Acetic acid ----- CH^O.^.
Leucein - - - C^H.^NO,.
Second alkaloid - - CToHor.N.,0-
Alkaloids i ^^^^^^^^ ^irvaiui^ - -
\ Do, copper compound - 0^2-^23^^^!^'^ 3^7-
\ Third alkaloid - -
Leucin - - - - - C^H^.^NO^.
Ditto co2:)per compound, mono- ) .^/q -j^q ^q^^
cupric dileucin- - - .\-\^vi 2/ •
Glycoleucin, isomer of leucin - C^.Hj3]SI02.
The cupric compound is also \
isomeric with the cupric com- > 2(C,;Hj2NO.,)Cu.
pound of leucin - - . j
Tyrosin ----- C9Hi,N03.
Tyrosin mercurous chloride i^er- ( jj ^.j,
curie oxyde - - - _ j v 9 10 .5 & / • o
With this li£t the number of products is by no means exhausted.
Only few quantations have as yet been made, so that the materials
for drawing any conclusion regarding the constitution of the albu-
minous substances of the brain are not yet to hand.
INTRODUCTION.
The inorganic ingredients or mineral substances of the brain are
distributed amongst its juices and solid ingredients in a very re-
markable manner, as will be discussed more fully below. These
bodies were formerly studied by means of analyses made on
materials obtained by combustion of the brain as a whole. This
proceeding had two sources of fallacy connected with it, which
greatly diminished the value of its results. In the first place, the
phosphoric acid produced by the destruction of the phosphatides
(in which, of course, phosphoric acid is in organic combination)
was calculated as mineral phosphoric acid, it being found partly
in combination with bases, partly free. Owing to its being in
excess over the whole of the saturating power of the bases, it ex-
pelled all volatile acids, such as carbonic, and sulphuric, and
chlorine, and was not even itself entirely preserved from the re-
ducing influence of the glowing charcoal, which volatilized a
portion of it as phosphorus. This inconvenience was only partially
avoided by the use during combustion of caustic baryta or barium
nitrate. In fact, by no method as yet proposed could the sulphur
or phosphorus in organic comUnation be kept separate from that in
inorganic combination. Some progress has, however, been made
by the separation of the soluble educts from the insoluble ones,
and from the interstitial juices ; and as each of these complex sub-
stances retains mineral matters peculiar to itself, while the phos-
13hatides can be almost completely excluded by precipitation with
acid from their solution or suspension in water, it is feasible to
obtain, by a minimum of three sets of analyses, some insight into
the nature and distribution of the mineral ingredients of the
brain. I enumerate the elements which enter into their com-
position :
Metals: sodium, potassium (ammonium), calcium, magnesium,
iron, manganese, copper.
Metalloids : chlorine, sulphur, phosphorus, carbon, oxygen,
hydrogen, fluorine.
There is met with at almost all steps of the separation of the
ingredients of white matter, a substance distinguished by fusibility
and insolubility in boiling alcohol, which was named stearoconote,
and regarding which a few explanatory notes may properly be
given in this place. When white matter is placed into a quantity
of hot alcohol, most cholesterin and lecithin and some cerebrin
dissolve ; but the other phosphorised and nitrogenised matters
'24: CHEMICAL CONSTITUTION OF THE BBAIN.
immediately fuse into a plaster-like mass, which is then practically
insoluble in boiling alcohol. There is therefore here an attraction
produced by the fused state which removes the solubility of some
of the ingredients of the mass in alcohol. When the principal
matters soluble in ether — i.e., the kephalins — are now extracted,
the fusibility is diminished, and some of the cerebrins dissolve
more freely in hot alcohol. Stearoconote, however, still forms,
and is now a reaction mainly of the myelins and the cerebrosides.
When all the myelins are separated from the cerebrin group of
bodies, their power of forming stearoconote is depressed to a
minimum, but not entirely removed. The myelins by themselves
also retain this tendency of forming a fused mass insoluble in
boiling alcohol, which in the text has been termed a tendency to
stearoconotise. The phenomenon seems to be compound, and not
simple, and to consist in essence in a dehydration without attendant
change in quantities of atoms of other elements. For T have found
that some stearoconote free from phosphorus could, by treat-
ment with benzol and a little hydrochloric acid, be transformed
into a soluble substance, which on elementary analysis was found
to have the same empirical composition as the stearoconote from
which it was made. In this case no separation of a base from the
substance could be proved to have taken place. In other cases it
was found that the stearoconote obtained its solubility in alcohol
without the intervention of acids, by standing in benzol, in which
it is easily soluble. In a third class of cases the stearoconote re-
gained its solubility in boiling spirit by prolonged treatment with
hot or boiling water. Again, in other cases it was probable that
the stearoconote was really a compound of some nitrogenised body
with a mineral base, and bodies resembling the stearoconotes in
some respects, but not in all, were produced by adding bases to
solutions of the cerebrin series. On the whole, then, ' stearo-
conote ' does not seem to be a chemical individual differing from
the other bodies described, but a function of several bodies, which
pass into that state by a molecular change, which change can be
made to retrograde mto the originally soluble condition.
The function is a result of chemical and physical influences
acting simultaneously (alcohol and heat), and is apparently not
obtained by either influence separatel}^ Stearoconote seems,
therefore, a product, and not an educt ; but it is indirectly im-
portant, as showing that there is some peculiar attraction or
INTRODUCTION.
25
mutual influence between the bodies of the two great groups. In
white matter where those brain-principles are all mixed together
nearly in their original proportions (only lecithin being much
diminished in quantit}^), the above influence has its highest in-
tensity. The function diminishes with increased separation — i.e.,
increased purity of educts. It is entirely lost in some of the
educts, when they are in the pure state ; thus pure kerasin could
by no means be made to yield any stearoconote. But it remains
with others, particularly myelin, paramyelin, and sphingomyelin
in the pure state, and is evoked when they are subjected to the
influence of heat in the presence of a quantity of alcohol insuffi-
cient to dissolve the whole of the material before it has had time
to fuse. Once fused, it seems to resist solution, even after re-
peated powdering to increase the surface for the action of alcohol,
to the extent of being rather destroyed than dissolved.
In the phenomenon just described we observe as the main result
diminished or suspended solubility — that is to say, suspension of
the lowest form of chemical attraction. In a phenomenon now to
be pointed out, we j^erceive however, on. the contrary, the produc-
tion or elicitation of a solvent power of a kind hitherto quite
unknown in animal chemistry. Some of the phosphorised bodies,
when combined with certain metals or metallic salts, in the pre-
sence of ethylic ether, and whether dissolved in it or not, cannot
be separated from these metals by hydrothion. The dissolved
compounds assume the colour of the respective sulphide, and re-
main dissolved ; the compounds insoluble in ether immediately
pass into solution when the hydrothion gas is passed through the
mixture. Thus, cadmium chloride compounds dissolve with a
canary-yellow colour ; platinum chloride compounds with a dark
brown colour ; lead compounds with a red or blackish-red colour.
The compounds can be partially or entirely precipitated by water,
or alcohol, or ammonia, etc. They contain, in addition to the
phos2:)horised body, in the cases of chlorides, chlorine and sulphur,
besides metallic sulphide ; in the case of metals like lead, metallic
sulphide and sulphur. Their complication places them at present
beyond the reach of stoichiometric treatment.
When once thus combined with sulphur (or hydrothion) and
metallic sulphide, or with these and chlorine in addition, and pre-
cipitated and isolated, the phosphorised bodies can by no means
ordinarily at hand be again obtained in the free state. The com-
26 CHEMICAL CONSTITUTION OF THE BRAIN
pounds with metals of the phosphorised bodies can be decomposed
by hydrothion only while suspended in water or spirit : the pro-
duct, a mixture of metallic sulphide with the respective body,
must then be extracted by a suitable solvent.
The obstacles which these extraordinary properties of the
chemical principles of the brain throw in the way of chemical
i:)rocedure are perfectly indescribable. To use a simile from
military life, the biologist who attacks his problem in front is
beaten off at all points ; he can only conquer it by flanking on
long and circuitous routes, and by the use of instruments of
warfare which are either new or superior to those hitherto in
use.
These difficulties increase with every step which leads nearer to
the consummation of the purpose, and are greatest at the point
where the isolated chemical individuals are to be freed from the
last traces of admixed impurity. The very peculiarities of the
principles described are mostly negations of the properties ordin-
arily relied upon as criteria of chemical purity. On this matter I
have repeatedly dilated .in the text, and given expression to
apprehensions as well as to considerations calculated to remove
them. As the difficulties arose they were followed out experi-
mentally, one by one, step by step, to the exhaustion of the
known means at hand, or of the new means that could be devised
in the time. But it must be left to the future to increase, if
possible, both the means of producing, and also the criteria for
insuring, that absolute purity of ultimate educts which is the con-
dition of certainty in chemical science.
It is therefore not asserted that the absolute limits of the
subject have anywhere been reached ; but it is confidently be-
lieved that its entirety has been explored in such a manner that
fundamental truths cannot have escaped observation, and that
what remains to be done is essentially of the character of detail,
which, however vast by multiplicity it may become, will not alter
the broad outlines which this investigation has led me to state.
While, then, there may be some degree of uncertainty as to the
absolute purity of some of the principles involved, there can be
none as to their striking individuality ; and as regards this, the
positive evidence of their peculiar and distinctive qualities is so
strong that the fact of their not uniformly answering to certain
other criteria is, in my opinion, quite insignificant.
INTRODUCTION.
21
By the researches embodied in this treatise the brain is shown
to be the most diversified chemical laboratory of the animal body ;
it is shown that all other organs, even when the results of their
chemical action, be they destined to take a centripetal or centri-
fugal course, are added to them, are relatively much more simple
and very much less specific in their chemical constitution than the
organs producing and conducting nerve-power.
28 CHEMICAL CONSTITUTION OF THE BBAIN.
II.
METHOD PURSUED FOR THE ISOLATION OF
IMMEDIATE PRINCIPLES.
Preparation and Coinminution of Bmintissue, — Anyone who
would i^roceed to an extensive chemical investigation of the brain
should procure, in the perfectly fresh state, and with the perfect
freedom from disease which are indispensable, such a supply of
material from the human subject as will suffice for his purpose.
Human brains are not only relatively the largest in size, but also
the richest in specific ingredients, and therefore the most advan-
tageous objects of inquiry. But they are, under ordinary circum-
stances, difficult to obtain, and therefore for large and more
general inquiries ox brains may be used. Five such brains weigh
on an average 1,780 g, or eight weigh six pounds. They are
washed once and freed from clotted blood. They are next care-
fully skinned, the arachnoid and pia mater being removed by
means of fine anatomical forceps. When the brains are entire
they may be skinned in the ordinary anatomical manner, which
employs two pairs of forceps, worked simultaneously and anta-
gonistically by both hands of the operator. When, however, the
brains are much broken up, each piece must be held in one hand
while being freed from membranes with the other. The skinned
parts are again rinsed in water, and then placed in water for a
short time ; next placed upon a sieve to drain, and then sub-
merged in methylated alcohol of 85 per cent, by weight in volume
strength, previously purified by distillation over tartaric acid.
Great care is necessary to supply a sufficient amount of alcohol,
so that the brains may be quickly dehydrated and hardened.
For if the alcohol is too dilute, or becomes too dilute l)y being
insufficient in quantity, the brains remain soft and unworkable,
ISOLATION OF IMMEDIATE PRINCIPLES. 29
and decompose with a fetid odour. For the same reason all
brains, before submersion in alcohol, must be broken up, or sliced
into small pieces, so that they can be easily penetrated by the
alcohol. This is required even when strong alcohol is used, as
this is liable to harden the outer shell merely, and leave the inside
of the brain to soften and decompose.
The washing and submersion in water probably remove small
quantities of extractives and soluble salts, besides the blood, and
must therefore be carried out with care. The solutions so ob-
tained are thrown away, but the alcoholic solutions in which the
brains have been hardened are purified from albumen by boiling
and filtration, freed from spirit by distillation, and evaporated to
the consistence of extracts on the water-bath. They are to be
considered as water-extracts, and are mostly free from specific
brain substances, and only yield extractives and salts and other
matters, which will be described in the relative chapters.
When the brains are well hardened in the alcohol, which fre-
quently requires the repeated removal of the watery spirit and
substitution of fresh strong spirit, they are passed through a
rotary mincing machine, and the minced portions are again mixed
up with strong alcohol. This pulp is now worked through a very
fine hair sieve, having 144 meshes to the square inch, and each
of the twelve strands of hair crossing the square inch in one direc-
tion, being composed of eight single horse hairs ; the sieve stands
upon a glass-receiver in such a manner that it cannot move, and
no matter which passes the sieve can be lost. The trituration on
the sieve is effected by a strong circular brush, which is rubbed
over the sieve by the hands of a workman. When passed
through the sieve the brain is in the state of a very fine pulp or
purree^ and is ready for extraction. All other modes of commi-
nution which have been recommended are less useful than the
foregoing ; they are either inefficient or slow and laborious ; in
particular, trituration in a mortar with a pestle is very inefficient.
All methods of comminution which do not reduce the brain to the
finest possible pulp, or smooth paste, must be rejected, as from
imperfectly comminuted brains the immediate principles are
necessarily most imperfectly extracted.
Extraction of Prainpulp and Separation of Albuminous or Insoluble
Matter, White Matter, Buttery Matter, Last Oihj Matter, and Ultimate
JFatery Mother- liquor. — The smooth paste of brain-matter is now
CHEMICAL CONSTITUTION OF THE BRAIN.
mixed Avith a considerable amount of alcohol of 85 per cent., and
heated in a well-tinned large sauce2:)an over a gas lamp by a star-
burner or any other heat source, while being stirred with a wooden
rod without intermission. When it has reached the temperature
of 70° the mixture is removed, and immediately poured on a
filtering cloth stretched and tied over the top of a large earthen-
ware jar or pan. The filter iy covered with a wooden cover.
When the liquid has percolated, the pulp is removed from the
cloth with a fiat spoon, and again placed in the saucepan, mixed
with spirit, heated to 70° while being stirred, and again placed
upon the same filter as before. This operation is repeated in all
about five times, when the brain-matter is exhausted of all matters
which alcohol at that temperature will dissolve. The matter is
now tied up in the cloth and pressed in a screw-press. It comes
out as a solid, somewhat elastic cake of alhum'mous or insoluUe
matter, of which the analysis and description will be given in a
future chapter. In my earlier experiments I arrested the heating
at 45°, because it has been so frequently stated that when brain-
matters are heated beyond they decompose. But since I know
the behaviour of the isolated brain-matters, I have come to con-
sider this statement as unfounded. Moreover, there is a consider-
able quantity of cerebrin-like matter in brain which is not at all
dissolved by spirit at 45°, but is taken up by boiling spirit, as
has been shown in Ann. Chem. Med. i., 1879, 258. To extract
all this matter it is necessary to boil the albuminous matter with
spirit for many hours in a platinum still, with a condenser attached,
and to repeat this at least from twelve to fourteen times. But
the low heat is certainly convenient, and need not therefore in
the earlier extraction be overstepped. For the same reason a
saucepan of cast-iron, about three gallons capacity, is preferable to
any other vessel of tin or glass. The earthenware jars or pans
should be of a capacity of from twelve to fifteen gallons. I have
found them most useful, and carried on all operations from the
soaking and hardening to the last filtration of the buttery preci-
pitate with their aid.
White Matter. — The alcoholic extracts are all united, and allowed
to cool during from twelve to twenty-four hours. In hot weather
the cooling must be assisted by placing the jar in a tul) and
surrounding it with cold water and ice. The extracts during
cooling deposit a large amount of v-hite cri/staliine and (jramdar
ISOLATION OF IMMEDIATE PRINCIPLES.
31
prec'qntate, which adheres to the sides and covers the bottom of
the vessel, while the alcohol is perfectly clear, though coloured
slightly yellow. The whole is filtered through a cloth stretched
over a pan, and when the entire precipitate is collected on the
cloth, and has been condensed by stirring with a spoon, the cloth
is removed, tied up, and placed in the screw-press, and all
mother-liquor thus removed. When taken out of the cloth the
precipitate presents itself as a hard white cake, which can be
broken into pieces, and constitutes the particular white matter of
Vauquelin, and will in the text be signalised as tvhife matter.
When the abbreviations fV.M. occur in the description of
any preparation, they indicate that the preparation has been
extracted from this white matter. I shall not describe this white
matter any further, nor have I instituted any experiments upon
it such as Vauquelin made, because it is evidently a very compli-
cated mixture, containing nearly the whole of the substances to
be described as the cerebrosides, stearoconotes, cholesterins,
kephalins, myelins, and lecithins, and small quantities of other
matters. The processes by which these substances may be ex-
tracted will be given lower down, after the description of the
treatment of the alcoholic extract has been completed. Here it
may yet be stated that the white matter can be jireserved in
stoppered bottles, in a cool place and protected from light, almost
unchanged for a very long time, In contact with absolute alcohol
it also remains unchanged, though gradually yielding a yellow
extract ; but in contact with ether it yields kephalin to the latter,
which is quickly oxidised into a red substance having a green
fluorescence ; this effect seems due to the peroxide of hydrogen
produced during the oxidation of the ether. I have therefore
limited the use of ether to the most necessaiy 02:>erations, and
then cause the substances to pass through these with great
despatch, so that this oxidising effect of the ether is as far as
possible avoided.
BiiUery Matter. — The Alcoholic Filtrate from the Wldte Matter
now placed in a capacious tinned copper, or better platinum still,
and a great part of the alcohol is distilled off. When a certain
degree of concentration has been obtained, which is determined
by experience, the hot liquid is thrown into a pan, and again
allowed to cool, assisted if necessary with cold water or ice. It
now deposits a second quantity of matter, which is less solid and
32 CHEMICAL CONSTITUTION OF TEE BBAIN.
more coloured than the first, and after filtration remains on the
cloth as a semi-solid plastic substance, to which I have given the
name of the hidterij matter. This can only be freed from mother-
liquor by manipulation with a spoon, and must not be pressed too
hard, as it is liable to pass through the meshes of the cloth. The
buttery matter consists of much chole&terin, lecithin, little myelin,
kephaloidin, and some cerebroside, and small quantities of other
matters ; the substances are therefore qualitatively mainly the
same as in the white matter, but they are present in entirely
different proportions. The buttery matter also keeps well in a
bottle by itself, or in the presence of alcohol, but should also not
be kept long in ether.
Last Oily Matter. — The Alcoholic Filtrate from the Buttery is again
distilled so long as good spirit passes over and no precipitate
ensues in the fluid. When these conditions are exhausted it is
placed in a large dish on a water-bath and evaporated. At a
certain period oily drops make their appearance, which adhere to
the sides or float in the fluid, and unite to larger round globular
masses. They separate easily while the fluid is hot, but w^hen
the fluid cools they swell, become flaky and distributed in the
fluid, and cannot be filtered. They are best separated while hot
by a separating funnel, to which they adhere, while the fluid sinks
down ; or they may be collected on a paper filter on a hot funnel.
This matter has received in my laboratory the title of the last
oily, hy which it will be signahsed in this essay. It consists
mainly of phosphorised bodies with little cholesterin, and some
peculiar not yet accurately defined matters.
Ultimate Watery Motherliqnor. — The Filtrate from Last 0//// con-
stitutes the ultimate icatei y mntherliquor and contains all matters
from the brain which are highly soluble in water, such as the
salts, the extractives, and soluble immediate principles to be
described. This liquid is evaporated on the water-bath to the
consistence of a thin extractive, and placed in bottles until further
examined as will be described. While the preparation accumu-
lates it is well to keep the extract covered with some absolute
alcohol to prevent the formation of mould on its surface.
Process for sejjarativg White Matter into its Constitvents. — The
following process, which for the purpose of alibrcviation I will
term ' ether process, W.M.,' yields the cerebrins very quickly and
directly, and leaves little myelin, some paramyelin, and most
ISOLATION OF IMMEDIATE PRINCIPLES. 33
sphingomyelin, with the cerebrins. It also yields kephalin, but
much remains in the mother-liquors. The myelin, lecithin, and
cholesterin remain in the ultimate mother-liquor, and can be
separated only by cadmium or platinum chloride.
The white matter is fully extracted with ether in stoppered
bottles, with the precaution of using the same ether for several
portions of white matter so as to obtain saturated solutions. All
secondary solutions and washings are concentrated by the still.
For this purpose French flasks and a platinum condenser are used ;
the flasks contain spirals of platinum-wire, and pieces of tobacco-
pipe tube strung on platinum-wire. This arrangement prevents
bumping. The whole of the solutions are now exposed in bottles
(stoppered) to a strong freezing-mixture of ice and salt, and the
clear coloured ether is quickly siphoned off the dense white
deposit. All siphons are of glass tube, with movable caoutchouc
joints, and mounted in corks, so that they can be applied to the
bottle to be emptied on one side and to the bottle to be filled
on the other side, air-tight, and be started either by blowing
(with the air-bellows) or by suction at the opposite end. The
inner limb of the siphon is so curved as to be near the side of the
bottle and easily visible ; its end is directed sideways to prevent
an upward rush of deposited matter.
This apparatus gives to the operator full power to start and
arrest the flow of the ether whenever he finds it desirable, and
enables him to regulate the suction-pipe, and so take off the last
portions of ether above the deposit, without losing much ether
or being molested by it. When the principal mother-liquor is
removed, pure ether is thrown upon the deposit, which is again
frozen. The deposit is much less soluble in pure ether than in
the mixture of dissolved matters, a peculiarity shown by almost
all brain-substances. The deposit, when dense, frequently forms
a firm cake at the bottom of the bottle, which comes off as a
round disk. These ^ fird deposits hy frost from ether extracts of white
matter ' are separated into sphingomyelin, myelin, and other
matters, as will be described lower down.
When the ether extracts give no further deposits on exposure
to renewed freezing-mixtures, they are treated with absolute
alcohol until all kephalin is precipitated. If the alcohol be
watery, even slightly, say of 80 to 90 per cent, strength, the
deposit contains much cholesterin, particularly if the ether
3
34 CHEMICAL CONSTITUTION OF THE BEAIN
solution is concentrated. Absolute alcohol should therefore
be always taken as well as absolute ether for these operations ;
many other reasons which will appear in the sequel support this
desideratum to the extent of making it an absolute condition of
perfect success.
The ether-alcohol mother-liquor is now distilled for removal of
the ether, and is then while hot treated with a hot solution of
lead acetate and ammonia as long as a precipitate is thereby
produced. The precipitate, which contains kephalin-lead,
myelin-lead, and a few other compounds, is filtered off. The
solution is allowed to cool, and deposits more lead [salts and
cholesterin. The filtered solution is again distilled while carbonic
acid is passed through it. By this means the excess of lead is
precipitated and the ammonia expelled. To the alcoholic filtrate
alcoholic cadmium chloride is now added as long as a precipitate
is produced, and afterwards a large excess of the same cadmium
chloride solution. The precipitate is filtered oflf on a cloth pressed
hard, and immediately placed in ether. A cadmium salt of a
phosphorised matter dissolves, which is not yet very well defined.
Cholesterin also dissolves. The greater bulk of the cadmium
chloride j)recipitate remains insoluble in ether, and consists of
lecithin, paramyelin, amidomyelin, and sphingomyelin, all com-
bined with the metallic chloride. These compounds can be
separated from each other, and the immediate principles can be
isolated from the compounds by processes to be described.
Treatment of the Butter?/ Matter. — The buttery matter is dissolved
in hot spirit, and completely precipitated with hot ammoniacal lead
acetate. The filtrate is allowed to get cool and deposit more lead
salt and cholesterin, to be separated hy ether. The united lead
salts, etc., after exhaustion by ether, are treated with spirit, etc.,
for the isolation of their ingredients, as will be described below.
From the main alcoholic filtrate the excess of lead and ammonia
are removed by a current of carbonic acid and distillation. To
the clear filtrate alcoholic cadmium chloride is added in large
excess, and the white precipitate is treated just as the precipitate
from white matter above described. Ether extracts from it
cholesterin and a phosphorised cadmium chloride compound, whiie
the part insoluble in ether is separated by the benzol process into
three or four compounds of lecithin, jjaramyelin, and amido-
myelin.
ISOLATION OF IMMEDIATE PRINCIPLES. 35
Treatment of the Last Oily Matter. — This is dissolved in hot alcohol
and precipitated by lead acetate and ammonia ; the precipitates
formed in the hot fluid, and after its cooling, are collected and sepa-
rated into their constituents by ether and spirit, etc., as will be
described. The spirit is freed from lead and ammonia by distil-
lation with the aid of a current of carbonic acid. The filtered
alcoholic solution is precipitated with an alcoholic solution of
cadmium chloride added in large excess, and the cadmium com-
pounds are collected and treated as will be described.
Summary of Immediate Principles in the crude state or Mixtures
thereof isolated. — We have thus separated the brain into the
following immediate principles or mixtures thereof :
1. First extractives (by soaking alcohol).
2. Insoluble albuminous residue.
3. White matter, containing —
{a) Kephalin (with varieties and compounds) ;
(b) Lecithin (with varieties and compounds) ;
(c) Paramyelin (with varieties and compounds) ;
(d) Myelin (with varieties and compounds);
{e) Amidomyelin (with varieties and compounds) ;
(/) Cholesterin and phrenosterin ;
{g) Cerebrin mixture, or mixture of cerebrosides, cere-
brinacides, cerebrosulphatides, and amidolipotides,
containing also sphingomyelin and assurin.
4. Buttery matter, containing —
{a) Kephaloidin (with varieties and compounds) ;
{h) Lecithin (with varieties and compounds) ;
(c) Paramyelin (with varieties and compounds) ;
{d) Myelin (with varieties and compounds) ;
(e) Amidomyelin (with varieties and compounds) ;
(/) Sphingomyelin, assurin, cholesterin and phreno-
sterin ;
{g) Cerebrin mixture (very small amount), amidolipotides.
5. Last oily matter —
{a) Lecithin ;
(&), Paramyelin ;
(c) Oily lipoid matters, cerebrols.
6. Ultimate watery extracts of brain, containing —
{a) Alkaloids ;
(h) Amidoacids and imides ;
(c) Carbohydrate ;
(d) Organic acids and salts ;
(e) Liorganic or mineral salts.
3—2
36 CHEMICAL CONSTITUTION OF THE BRAIN.
Summary and Arrangement in Groups of Immediate Principles. —
In the foregoing descriptions of processes I have, for the purpose of
brevity, spoken of the crude immediate principles which were
isolated as if they consisted of a single body each ; but in the
summary list just given I have added to most of the names of
these compounds an enlarged definition, by which not only the
2mre princi])le, but also varieties of it and comjmmds of it are said
to have been isolated in one and the same operation. This is
strictly the case, as will be seen, for example, in the account of
kephahn hereafter to be given ; but it may be at once stated that
the pure immediate principle which gives rise to the name con-
stitutes the great bulk of each preparation, that this bulk is
relatively greatly increased by the removal during the process of
purification of matters in chemical combination with a smaller
portion of the immediate principle, and that the varieties are very
similar in type to the principal matter, and whenever they
cannot be separated absolutely their nature and quantity can
be ascertained by very good approximations. Where matters are
so similar in i3roperties as kephalin and kephaloidin, a complete
separation of the entire quantity of each from the other is not
easily eff'ected, but a quantity of each can be obtained pure from
the other, and from the results obtained by their chemical study
the composition of mixtures in any proportions can be derived.
The immediate principles above enumerated may be arranged in
the following groups :
Group of Phosphorised Principles or Phosphatides.
Subgroup of Monoidtrogenised Monophosphatides (N : P = 1 : 1) :
Lecithins
Kephalins ;
Paramyelins;
Myelins.
(Sphingomyelic acid, a product.)
Suhgroiip of Dinitrogenised Monophosphatides (N : P = 2 : 1) :
Amidomyelins ;
Amidokephalins ;
Sphingomyelins (Apomyelins).
Subgroup of Dinitrogenised Dipkosphatide (N : P = 2 : 2) :
Assurin.
ISOLATION OF IMMEDIATE PRINCIPLES.
37
Subgroup of Nitrogenised Phosphatide-sulpliatide :
Cerebrosulphatide, body from group of cerebrinacides, con-
taining probably phosphorus, nitrogen, and sulphur (?).
Subgroup of Nonnitrogenised Monopliosphatides :
First acid from buttery matter, lipophosphoric acid.
Second acid from buttery matter, butophosphoric acid.
(Kephalophosphoric acid, a product.)
Group of Nitrogenised Nonphosphorised Principles.
Subgroup of Cerebrosides :
Phrenosin ;
Kerasin.
Subgroup of Cerebrinacides :
Cerebrinic acid ;
Sphaerocerebrin ;
Other principles not yet defined.
Subgroup of Cerebrosulphatides :
Body containing sulphur.
Subgroup of Amidolipotides, or nitrogenised fats :
Bregenin ;
Krinosin.
Subgroup of Alkaloids :
Hypoxanthin ;
Second alkaloid, gladiolin ;
Third alkaloid, tennysin.
Subgroup of Amidoacids and Amides :
Leucin and homologous principles ;
Tyrosin ;
Urea.
Group of Principles composed of Three Elements only.
Subgroup of Alcohols, nonnitrogenised :
Cholesterin ;
Phrenosterin (?).
Subgroup of Carbohydrates :
Inosite ;
Glycogen (?).
Subgroup of Organic A cids, nonnitrogenised :
Formic acid ;
Sarcolactic acid ;
Succinic acid ;
Oxyglyceric acid.
38 CHEMICAL CONSTITUTION OF THE BB JIN
Group of Albuminous Substances.
Subgroup of Nitrogenised Sulphatide-pliospliatkles :
Plastin ;
Gangliocytin, Cytophosphatide (aNuclein).
Stihgroup of Nitrogenised Sid/phatides :
Albumen ;
Collagen.
Grou^ of Inorganic Frinciples, including both acids and bases,
and salts, either free or in combination with many of the fore-
going organic principles. This comprises :
Sulphuric acid ;
Hydrochloric acid, and chlorine in chlorides ;
Phosphoric acid ;
Carbonic acid ;
Potash \
Soda I In combination with immediate principles,
Ammonia I forming their bases, or in combination with
Lime \ phosphoric acid, and attached to immediate
Magnesia t principles as phosphates, or in combination
Copper I with mineral acids, as free mineral salts in
Iron I the juices and extracts.
Manganese J
Alumina, silica, fluorine (doubtful).
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 39
III.
GROUP OF PHOSPHORISED PRINCIPLES OR
PHOSPHATIDES.
General Properties of the Phosphorised Principles. — This group
comprises kephalin, kephaloidin, myelin, sphingomyelin, assurin,
lecithin, paramyelin, amidomyelin, and their congeners and com-
pounds. All these bodies have certain properties in common,
which point to some similarity in chemical constitution ; by other
peculiarities, again, they are sharply distinguished from each
othei". Some are soluble in water in a certain manner and measure.
When they are in the dry state, and are placed in pure water,
they sink to the bottom, and are at once wetted by the water.
Thus their specific gravity is shown to be greater than that of
water, and by this, and the faculty of being wetted, they are
sharply distinguished from the fats or fatty acids, but assimilated
to the soaps, as the older authors correctly stated. When they
have remained in the water for a short time they begin to swell,
to become transparent at the thin margins of the particles, and
covered with a loose layer all over their surface. This on agita-
tion is easily detached, and floats in clouds in the liquid; the
clouds diffuse themselves indefinitely throughout the whole of the
water, and if enough water is present, the mixture is frequently
shaken, and some time is given, the particles of solid matter dis-
appear entirely and form a turbid solution of very peculiar appear-
ance. As will be seen hereafter in particular, these solutions can
by no mechanical means be clarified ; yet they are solutions, and
pass through many layers of the finest Swedish filtering-paper.
Most particles are so small as to be beyond the reach of optical
definition as single particles. They exhibit their presence, how-
ever, by iridescence in the case of myelin and lecithin, and by
40 CHEMICAL CONSTITUTION OF THE BE A IN
reflecting polarised light in all cases. These solutions, therefore,
resemble somewhat the cold solutions of soaps, and the emulsions
produced by solid fatty acids with neutral phosphates, the emul-
sion produced by vegetable seeds (almonds) with water, etc. ; but
they differ from milk and emulsion of fats and gum by there being
few particles visible. Moreover, emulsions are supposed to require
the presence of two agents besides water, whereas these brain
substances give this peculiar solution with mere water ; they are not
decomposed, as the soaps are, into acid and alkaline salt, as they
are not salts, but form these solutions in the free and uncombined,
but also in the combined state. I can give no better definition of
this peculiar condition than by describing it as a state of imperfect
or incomplete solution, a stage intermediate between the solid and
the fluid state of matter. Those who do not coincide in this de-
scription may term the solution an 'emulsion,' if that conveys any
definite idea, or a state of the finest subdivision of particles, with
peculiar attraction of these particles to water, and consequent re-
pulsion of particles from each other. This latter part of the
question I shall have to consider at greater length when I come
to discuss the dependence of the structure of the brain upon the
chemical and physical characters of its ingredients. For the pre-
sent purpose it suffices to sum up that these phosphorised bodies
have all an extreme attraction for water, swell in it, and ulti-
mately form a nearly perfect solution. Amidomyelin, when
obtained by dialysis from cadmium chloride salt, forms a perfect
solution, which filters like water, but congeals by warmth.
Sphingomyelin, when quite pure, contracts again and then sinks
in water, and can be filtered out of it. By this means they afford
good opportunities for mechanical and chemical purification now
to be described.
Deposition of Impnrities. — The solutions, on standing, deposit
any mechanical impurities, and any cholesterin contained in the
matters dissolving crystallises out and sinks to the bottom. Any
excess of the matters beyond saturation is also deposited, as well
as the less soluble compounds — e.g., sphingomyelin. If any cere-
brin were with the matters, it would also deposit. All solutions,
tlierefore, produced as above, and which experience has shown me
should not contain above 1 per cent, of the matters, must be
allowed to stand for a day or two, in order that these insoluble
impurities and admixtures may be deposited. The pure solution
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 41
is then removed with the siphon from the deposit, the solution is
filtered, and the deposit further exhausted with water.
Filtration of the Watery Solutions of the Plmphorised Matters. — -
When the solutions are placed on an ordinary filter of paper, a
portion passes by gravitation ; but the pores of the paper gradu-
ally become obstructed, and filtration ceases. It is therefore
necessary to expedite filtration by the aid of pressure. For this
purpose I have constructed a new apparatus, in which a vacuum
draws the liquid to be filtered into a hollow cylinder surrounded
with paper.
The filter is a hollow cylinder of silver-plate pierced like a fine
sieve, and covered with a six-fold roll of Swedish or Rhenish
filtering- j)aper, made secure at the top and bottom by a ring of
string, which also runs spirally over the whole j^aper. This
stands in a glass cylinder, which contains the fluid to be filtered,
and is always kept full by a perpetual siphon, drawing the
liquid from the reserve bottle ; as frequently as the fluid in the
wide upper part of the cylinder sinks below the oblique end of
the air-tube, which is wide and provided with a bulb, air is
admitted, and the siphon acts. The bottle receiving the filtrate
is evacuated of air gradually by means of the air-pump. The
tube which draws the filtered liquid from the cavity of the
cylinder goes to the bottom of the cylinder, so that nearly the
whole of the filtered liquid can at any time be drawn into the
receiver.
Such a cylinder, about a foot long and four inches in cir-
cumference, will, with a pressure of 70 centimetres of mercury,
filter a Winchester quart full of 1 per cent, solution of ]3hos-
phorised matter per day : the more, the purer the substance is ;
the less, the more cholesterin it contains. Sometimes only 1 00 cc.
are filtered per hour. In such a case a coarser filtering-paper
should be rolled over the Swedish, to collect the coarsest particles
and prevent them from getting upon the Swedish paper. After
some hours filtration becomes slow, and ultimately ceases entirely ;
then the paper is found covered with a gelatinous mass of undis-
solved matter and impurities. These may be rinsed off and again
extracted and filtered, but I have mostly found the matter so
small in quantity that I have discharged it with the paper. Such
a filter, with two changes of paper per day, may be going night
and day, and, if all corks are air-tight, which is easily eff"ected by
42
CHEMICAL CONSTITUTION OF THE BRAIN.
applying hot paraffin to them, requires very little attention except
a few strokes of the pnmp from time to time. When the sub-
stances previous to their solution in water were very pure, they
left no vestige of cholesterin crystals or of cerebrosides on the
filter. The filtered fluids are still opaque, those from kephalin
more coloured, those from myelin and lecithin of a milky white-
ness with the blue iridescence. When a good filtration had been
effected, a second filtration effected no improvement, and was
quickly accomplished. In this manner all phosphorised matters
used for cardinal preparations were passed through the process of
watery solution and filtration.
The process offers peculiar advantages for each of the three
varieties of phosphorised principles. Kephalin scarcely ever re-
tains any cholesterin, but myelin always does, unless it has passed
through the PtCl^, or lead process.
SoluhUUy of Phosphorised Matters in Ether. — Kephalin dissolves
in this reagent in almost any quantity ; lecithin only less than
kephalin ; but myelin is very little soluble in cold ether, more in
hot ether, and is instantaneously deposited from the ether as it
cools. Sphingomyelin behaves in a similar manner.
Sohihility of Phosphorised Matters in Alcohol. — Lecithin dissolves
in all proportions in hot absolute alcohol, less in cold ; kephalin is
almost insoluble in cold alcohol, more soluble in hot, almost en-
tirely deposited on cooling; myelin, paramyelin, amidomyelin,
and sphingomyelin are, however, little soluble in cold alcohol,
largely soluble in hot, and deposited on cooling in a crystallised
state, and in such quantities that the fluid becomes filled with
crystals. It will thus be seen that, while water offers no means
for the separation of these substances from each other, ether and
alcohol offer great advantages, which have indeed been utilised
in the construction of the method for their separation above
described.
A. SUBGROUP OF MONONITROGENISED MONOPHOSPHA-
TIDES. N : P = l : 1.
Lecithins.
Definition. — Lecithins are phosphorised and nitrogenised imme-
diate principles of brain-tissue and egg-yelks. There are three
varieties known, all containing neuryl, oleyl, glyceryl, and a third
PHOSFHORISED PRINCIPLES OR PHOSPHATIDES. 43
.acid radicle replacing hydro xyl in the phosphoric acid ; the three
varieties differ as regards the third acid radicle.
Isolation. — Lecithins can be isolated by the following process :
The alcoholic extract of brain-matter from which white matter
has been deposited is concentrated until on cooling it forms the
deposit called buttery. This is isolated by filtration, re-dissolved
in warm spirit and treated with lead acetate and ammonia as long
as a precipitate is produced, and filtered hot. The filtrate depo-
sits a crystalline mixture of cholesterin, lead salts, and lecithin.
This mixture is again isolated by filtration, dissolved in hot
spirit, and allowed to crystallise again. To the solution filtered
from the first deposit as well as to that filtered from the second
crystals an alcoholic solution of cadmium chloride is now added,
as long as a precipitate is produced : then half the volume of the
cadmium chloride solution already used is added to the mixture.
The precipitate is isolated by filtration and dissolved in boiling
spirit ; the solution, filtered hot, is allowed to deposit the salt,
which is then filtered off. This is a mixture of three, maybe
four, compounds, one of lecithin with cadmium chloride, another
of paramyelin with the same salt, a third of amidomyelin, and a
fourth of sphingomyelin with cadmium chloride. The last is, if
at all, present in the smallest quantity ; the lecithin and ami-
domyelin compounds are present in about equal quantities, and
constitute the grtat bulk of the precipitate. The precipitate is
dried in vacuo over sulphuric acid (when dried on the water-bath
it becomes coloured), finely powdered and exhausted with ether.
It is again dried and powdered, and treated for the separation of
the several compounds as follows :
The dry precipitate is suspended in a large volume of anhydrous
benzol and frequently stirred ; kephalin cadmium chloride and
kephaloidin cadmium chloride are extracted at this stage ; the
mixture is then boiled for some time in a water-bath, and a por-
tion of the benzol is removed by distillation. The mixture is
now allowed to stand in a cold place for twenty-four hours, and
then thrown on a filter. The matter remaining on the filter is
subjected to the same treatment with fresh benzol as often as
may be necessary to exhaust it of all matter soluble in cold benzol.
The matter insoluble in cold benzol is now again boiled with
benzol and ' thrown on a filter kept hot by a steam-jacket. A
benzol solution now passes through the filter, which deposits a
44 CHEMICAL CONSTITUTION OF THE BBAIN
compound on cooling, and keeps little or nothing in solution.
This treatment also is repeated until the insoluble compound
yields nothing to boiling benzol. The cadmium chloride precipi-
tate has thus been separated (leaving the kephalin compounds out
of consideration for the present) into three different matters :
(1) Compound soluble in cold benzol^ lecithin cadmium chlo-
ride.
(2) Compound soluble in hot benzol, deposited on cooling,
paramyelin cadmium chloride.
(3) Compound insoluble in benzol, cold or boiling, amidomyelin
dicadmium chloride.
As sphingomyelin is somewhat soluble in cold alcohol, it is to
be expected that a small quantity is present in the mixture of
which the three compounds just defined are the principal con-
stituents. It may be separated from the amidomyelin in the
manner to be described below. But its presence in the cadmium
chloride precipitate from buttery matter has not been proved ; in
the process of separating the educts by solvents only it remains
mainly if not entirely with the cerebrosides and cerebrinacides,
after the lead process with the cerebrosides only, to one of which,
kerasin, it has a slight chemical affinity. This might explain its
absence from the buttery matter.
Ejnsode concerning the Shifting of Cadmium Chloride in Mixtures
of Lecithin, Parcimyelin, and Amidomyelin, during recrystcdlisation
from Spirit. — It was observed that precipitates which had been
exhausted with ether, and therefore were free from uncombined
lecithin, after recrystallisation from spirit left some free lecithin
in the mother-liquor, which could be precipitated by renewed
addition of cadmium chloride solution. It had also been observed
that such preci^^itates containing amidomyelin showed a higher
percentage of cadmium chloride than before. Similar data were
also obtained during the treatment and analyses of the platinum
salts. On the basis of these observations the following hypothesis
was formed.
Amidomyelin probably combines with cadmium chloride in two
proportions, namely, either wdth one molecle of this salt, or with
two molecles, thus :
C,4H^,N.^P0iy + CdCl.^, containing 17-90 per cent. CdCl^.
C,^Hj,^^N,POi(>-f 2CdCl,, containing 30-37 per cent. CdCU.
PHOSPHOFJSED PRINCIPLES OR PHOSPHATIDES. U
The former has a tendency to saturate itself in the pi'esence of
lecithin cadmium chloride, C42Hg^NP09 + CdCl^, containing 19 "42
per cent. CdCl^, and during recrystallisation from spirit, or during
suspension in benzol in which the lecithin salt is in solution, takes
up some cadmium chloride from a portion of the lecithin, which
is thereby set free and remains in the spirituous, or as the case
may be, benzol mother-liquor. This kind of shifting of the cad-
mium chloride is more likely to occur in anhydrous solvents, or
strong spirit, whereas the dissociation under the influence of water,
complete during the process of dialysis, is partial, particularly in
dilute solvents or edulcorants.
Continuation of the Description of the Process. — The solutions in
cold benzol obtained in the process described in the foregoing are
allowed to stand for some days in the cold and repeatedly filtered.
They are then concentrated, allowed to stand, and filtered again,
until the residual solution remains quite clear and free from
deposit. Filtration is absolutely required to prove the absence of
deposits, as these are so transparent that they easily escape from
observation by the eye.
The concentrated thick benzol solution, which is mostly coloured
and strongly fluorescent, may now be evaporated to dryness, and
the residue further studied. But it is j)referable to add to it
absolute alcohol as long as a precipitate ensues. Time is again
wanted to complete the precipitation. The precipitate is washed
with alcohol, dried in vacuo, exhausted with ether, and recrys-
tallised from spirit. Now a portion remains insoluble in boiling
spirit, but the bulk dissolves and is deposited on cooling as a
white mass, which, under the microscope, is seen to consist entirely
of needles arranged radially in stars and balls.
Separation of Lecithin from Amiclomyelin and Paramyelin ivhen all
are in the free state. — All three bodies are precipitated by cadmium
chloride, and the compounds are soluble in boiling spirit. When
the mixture is decomposed in one way or another, and the result-
ing free bodies are dissolved in hot spirit, the amidomyelin and
paramyelin separate first in the shape of white leaflets, again on
concentration ; from the highly concentrated spirit a mixture of
lecithin and little amido- or paramyelin is at last deposited as an
unctuous mass. When this is treated with ether, white amido- or
paramyelin remains insoluble. When the ether solution is dis-
tilled to dryness and the residue treated with absohtfe alcohol,
46 CHEMICAL CONSTITUTION OF THE BRAIN.
lecithin dissolves, while again some amido- or paramyelin remains
insoluble. The absolute alcohol solution deposits more amido- or
paramyelin on long standing, particularly at low temperatures.
But it is, perhaps, not practicable to obtain perfectly pure lecithin
without a trace of amido- or paramyelin by this process, although
the amido- or paramyelin obtained by it is perfectly free from
lecithin. A specimen of lecithin thus prepared (by dialysis, etc.,
but not separated as CdCl2 salt by benzol) gave on analysis 3-66
per cent, P. and 2*83 per cent. N. The amount of nitrogen
shows that the body contained yet some amidomyelin.
The lecithin, when thus purified as far as j)Ossible, gives a white
CdCl2 salt which is soluble in boiling spirit, and when deposited
from it leaves some coloured impurity in solution.
On the mode of separating lecithin from amidomyelin by benzol,
when both bodies are combined with CdCl2, see ante^ and in the
relative paragraph under amidomyelin. Paramyelin can be sepa-
rated to some extent from amidomyelin by the greater solubility
of its hydrochlorate, but completely only by the solubility of its
cadmium chloride compound in boiling benzol, in which amido-
myelin is insoluble.
Properties of Lecithin. — Lecithin is a white crystalline body,
crystallising in thin plates, which when compressed form a wax-
like mass. It is very soluble in spirit, being only deposited when
the solution is extremely concentrated. A slight rise in the tem-
perature causes the crystals to redissolve. When to its cold
' saturated solution in spirit water is gradually added until a per-
manent considerable turbidity is produced, and when this turbidity
is cleared up by heating, the solution deposits on standing lecithin
in the semi solid hydrated state. The solution in 80 per cent,
spirit treated as descril^ed left 3*08 per cent, lecithin on evapora-
tion. The unctuous lecithin under the microscope consists of
balls in concentric layers. Lecithin is easily soluble in ether.
It is easily soluble in chloroform, and is left on evaporation as
a non-crystalline mass.
It dissolves in oil of vitriol with a yellow colour, and on addi-
tion of thick cane-sugar syrup Easpail's reaction, a purple colour,
is gradually produced, changing slowly into black. The reaction
is due to the oleyl radicle contained in the lecithin.
Comjmmds of Lecithin. — An alcoholic solution of lecithin is pre-
cipitated by cadmium chloride ; the white voluminous precipitate.
PHOSPHORISED PPJNCIPLES OK PHOSPHATIDES. 47
amorphous at first, crystallising on standing in the mother-liquor,
is easily soluble in boiling spirit, and dej^osited on rapid cooling
in white crystalline granules ; on slow cooling, acicular crystals
arranged in balls and rosettes are deposited. They are very
uniform and characteristic, and totally different from sphingo-
myelin cadmium chloride. This precipitate is insoluble in cold
and boiling ether, easily soluble in benzol.
Platinum Chloride Hydrochlorate of Lecithin. — Lecithin is preci-
pitated by an acidified solution of platinum chloride in spirit ;
the voluminous yellow precipitate is easily soluble in ether, and
again precipitated from this solution by absolute alcohol. Its
formula is 2(C43Hg4NP08)-f-2HCl + PtCl4. A compound also
occurs with one HCl only, and another without any HCl, and
containing PtCl^ only.
Lecithin is not precipitated by a mixture of lead acetate and
ammonia.
Solubility of Lecithin CdCl.^ Salt in Spirit. — Ten cc. of the 84 per
cent, spirit solution which had deposited a crystallised salt, on
evaporation left 0*0387 g. solid matter. One part of the salt
therefore requires 258 parts of spirit at 17° C. for solution.
Bearing of the Lecithin CdCl.^ Salt with Benzol. — When tlie crys-
tallised salt is placed in pure dry benzol it swells and becomes
transparent, but does not dissolve. If now the mixture be heated
for some time, the salt dissolves without residue, and the clear
colourless solution does not form any deposit on cooling and
standing. This j)henomenon is due to hydration of the crystal-
lised salt; during the first boiling with benzol the water is
evolved and passes over with the benzol vapours ; when clear
benzol passes over, all salt is and remains in solution.
Lecithin Hydrochlorate, C^gHg^ISTPOg + HCl. — The pure white
cadmium chloride compound is suspended in spirit, and the mix-
ture is saturated cold with hydrothion. It is now heated in a
water-bath to boiling while the current of the sulphuretted gas
is continued, until the filtrate is no longer affected by it. The
decomposed matter is thrown on a filter arranged on a steam-
jacket, and the colourless spirit solution of lecithin hydrochlorate
is separated from the cadmium sulphide. The solution deposits
the hydrochlorate as a felted mass of crystals. By slow crystal-
lisation from dilute solutions white crystals are obtained. As the
cadmium chloride yields two molecles of hydrochloric acid, of
48 CHEMICAL CONSTITUTION OF THE BRAIN
which only one combines with the lecithin, the mother-liquor con-
tains free acid in solution. As this acid decomposes lecithin at
high temperatures, it is not advisable to endeavour to obtain the
lecithin hydrochlorate which remains in solution in the spirit by
evaporation of the latter by heat. It is preferable to extract all
hydrochloric acid from the solution by mercuramin, reprecipitate
the lecithin by cadmium chloride, and redecompose this by
hydrothion in a minimum of spirit. If it is desired to obtain the
whole amount of lecithin in a given preparation as hydrochlorate,
the body should be neutralised with the acid, and the solution
evaporated in a vacuum over lime and oil of vitriol.
The hydrochlorate crystallises in thin leaflets, to be seen by the
microscope ; they are hexagonal, frequently saucer-shaped, and,
owing to their extreme tenuity, mostly so distorted and crumpled
up that they appear as a confused mass of curved needles.
{Human) Lecithin Hydrochlorate. — By CdCl2 from alcoholic solu-
tion of white matter and buttery. Passed through benzol process;
the soluble in the cold part again recrystallised from spirit. The
white salt was decomposed by hydrothion, the solution allowed
to crystallise ; the crystallised mass was recrystallised. Dried in
vacuo to a perfectly white mass which could be powdered easily.
At 98° it became a little soft, and by prolonged heating somewhat
coloured. It gave on analysis 4*84 per cent. HCl, 2-03 per cent. X,
and 4-29 per cent. P ; therefore, HCl : N : P = 1 : 1 : 1.
(Ox) Lecithin Cadmium Chloride.
Theory.
Atoms.
Per cents.
Found.
43 C
516
53-9
84 H
84
1
1 N
14
1-46
1-34 —
1 P
31
3-24
— 3-2i
8 0
128
1 Cd
112
11-71
2 CI
71
7-42
956
Lecithin
773
80-87
Cd CL,
183
19-13
956
C.^Hg.NPO.-fCdClo, At. W. = 942, requires 19-42 per cent, CdCl^.
LONDON
SCHOOL OF MEDICINE
fORWOMEK
PHOSPHORISED PPdNCIPLES OR PHOSPHATIDES. 49
Chemolysis of Lecithin. — Lecithin, when isolated as platinic
chloride hydrochlorate salt, immediately after isolation begins to
decompose, and completes this decomposition during the steps
necessary for its purification. Its platinum salt, which is in the
first instance soluble in ether, becomes in the air or in the vacuum
speedily covered with oily drops (oleic acid), and the residual salt
is then insoluble in ether. It was upon such changed platinic salt
insoluble in ether that the following chemolysis was eff'ected :
Water alone was found capable of removing at least some of
the platinic chloride ; but this treatment was not persisted in, and
the salt was boiled with two molecles of BaH202 during two hours.
The decomposition was eff'ected very readily, a black precipitate
(of platinum) being formed simultaneously and insoluble barium
salts of certain fatty acids.
The Barium Salts. — These were washed with water, and then
decomposed by hydrochloric acid, in the presence of ether, f \ The
ether solution was distilled to dryness, and the residual matter
converted into ammonium soap ; and the latter in its turn was
converted into barium salt once more, and after drying extracted
with much boiling absolute alcohol. The alcoholic extraction was
continued to perfection. The extracts deposited a white salt on
cooling, which was isolated, dried, and analysed; it gave 19-55
j)er cent. Ba. Oleate of barium requires 19-59 per cent. Ba. This
salt was therefore oleale of barium.
The barium salt left undissolved by the alcohol was insoluble
in ether. It was once more decomposed by hydrochloric acid in
the presence of ether, and converted first into ammonium soap,
and again into barium salt. It was now isolated, dried at 100° C,
and analysed; it gave 19-82 per cent. Ba, and 57-76 -per cent. C
and 9-86 per cent. H.
Computation of Analyses :
Percentages.
4- At. Wgts.
Ba = l.
0
57-76
4-813
33-4
H
9-86 ■
9-86
68-4
Ba
19-82
•144
1-0
0
12-56
•785
5-4
= CJg^Hg^BaO^.
This was, therefore, apparently margarate of harium.
The Barita Solution. — The excess of barita was first removed
from the solution by means of carbonic acid, and then nitric acid
4
50 CHEMICAL CONSTITUTION OF THE BRAIN.
was added to strong acidity. In this state the solution was pre-
cipitated by phosphomolybdic acid, and the filtrate reserved. The
precipitate was decomposed by hot barita and concentrated to a
low bulk. The barium which still remained in combination with
the base was carefully removed by the exact amount of sulphuric
acid necessary, and after concentration the resulting solution was
neutralised by HCl and precipitated by alcoholic PtCl^. The
platinic compound crystallised from water in long prismatic
needles.
The quantity of the platinum salt obtained was very near to
that which should have been obtained if all the nitrogen had
existed in one form in lecithin, and had been obtained in one
form.
Computation of Analyses:
Percentages.
-^-At. Wgts.
--Pt-i.
c
18-92
1-576
9-78
H
4-68
4-68
29-00
N
4-63
•330
2-00
0
5-61
•350
2-10
Pt
31-79
•161
1-00
CI
34-37
•968
6-00
= {C5H13NO), (HCl), PtCl,.
The filtrate separated from the phosphomolybdic acid precipi-
tate of the base was found to contain glycerophosphoric acid.
The chemolysis of lecithin, therefore, is complete, as there were
obtained :
Oleic acid - - - = C^gH^^
Margaric acid - - ^C^-H.^^ 0,
Glycerophosphoric acid = C3 Hg PO^^
J^eurin - - - =C, H^^N O
Total- - - =C43H9oNPOii
Deduct - - - O3 entered in chemolysis
Leaves - - = C^^.^H^^NPOg = lecithin.
I have lately chemolysed a specimen of lecithin which had
been as CdCl, salt entirely soluble in cold benzol, and had been
crystallised from spirit, after several earlier crystallisations had
been removed. It yielded oleic acid, margaric acid, glycerophos-
phoric acid, and neurin. In the course of this chemolysis I
observed that benzol is a better medium than ether for separating
PHOSPHOPJSED PRINCIPLES OR PHOSPHATIDES. 51
the lead oleate from the lead margarate. This lead process also
yields a purer margarate than the exhaustion with alcohol adopted
above ; but the oleate is again less pure than that deposited from
alcohol.
Note on Oleic Acid and its Reaction tvith Oil of Vitriol and Sugar. —
Oleic acid is generally considered to have much affinity for oxigen,
so as greatly to impede attempts to obtain it in a pure state.
This absorption of oxigen is considered and stated to be attended
with the production of a brown colour, and a change in the nature
and properties of the acid.
Pure oleic acid, prepared expressly from oil of almonds, with
the view of comparing it with the oleic acid furnished by lecithin,
showed none of these properties. The potassium, ammonium,
lead, and barium salts were all white, and showed no tendency to
become coloured on exposure. The free acid was also free from
this tendency, and was only very faintly yellow, while its solution
in ether was colourless and did not become brown on exposure.
To prove the purity of the body the barium salt was analysed for
barium, and found to contain 19"73 per cent, as against 19 "60 per
cent, required by theory.
Sulphuric acid turned oleic acid yellowish and red, but chloro-
form had no action on the mixture. It gave a solution with acetic
acid which was so turbid that no spectrum could be obtained.
But with sulphuric acid and sugar it immediately turned
purple (with sulphuric acid alone it was yellowish-brown), and the
product dissolved with a splendid purple colour in acetic acid ;
the solution presented the following spectrum :
In the concentrated state it passed red at A, thence a shade
increased to D, then black. A more diluted solution presented
one broad absorption band between D and E. End at G.
The edges of this band shaded off so very gradually that it was
difficult to fix the margins ; this solution was red, but presented
a green fluorescence ; it was not brilliant, and became more turbid
on standing.
Oleic acid in chloroform was now mixed with a drop of sugar
syrup, and then with sulphuric acid ; it immediately became in-
tensely yellow ; on stirring and breathing upon it, it became
purple, but the colour was very dark and mixed with brown.
It was soluble in glacial acetic acid, giving the above spectrum.
Chloroform extracted a splendidly purple matter, leaving a dingy
4—2
52 CHEMICAL CONSTITUTION OF THE BE A IN
one behind. This sokition, suitably dihited and kept anhydrous
by sulphuric acid, passed red to C. Further diluted, there was a
band between C and D, and another between D and G ; blue to C.
Again diluted, band 1 disappeared and band 2 contracted.
Acetic acid solution of same test gave same band. The band
became shaded off near green. Therefore with chloroform it
presented the same spectrum as phrenosin and kerasin, but these
were insoluble in acetic acid, in which the oleic acid test was soluble.
Theory of Lecithins considered as Phosphatides.
Oleo-Palraito-Glycero-Neuro-Phosphatide.
.0,
OP 4
^18^33^2
C3H.O3
Oleo-Margaro-Glycero-Neuro-Phosphatide.
f C*'i8^^3302 1
] C3H.O3
C,3H,,NP0,.
Oleo-Stearo-Glycero-Neuro-Phosphatide.
OP^
^18^35^2 L =n H IVPO
J
c
42
504
43
516
44
528
H
82
82
84
84
86
86
N
1
14
1
14
1
14
P
1
31
408 1
31
1
31
0
8
128
8
128
8
128
759
773
787
The first three radicles in each formula replace a molecle of
hydroxyl each ; but the addition of neurin takes place under cir-
cumstances which engender the expulsion of a molecle of water
from the neurin itself. The mobility of this molecle of water in
neurin is clearly shown in the course of the synthesis of this base.
2. Kephalins.
a. Kephalin.
Purification. — The crude kephalin obtained by the primary
operations is dried under the air-pump over sulphuric acid. It
PEOSPEORISED PRINCIPLES OR PEQSPEATIEES. 53
must be repeatedly taken out and flattened out in a mortar, and
iigain dried before it becomes dry and brittle, and can be
powdered. Resolution in absolute ether, reprecipitation by
absolute alcohol, and redrying causes a great improvement, but
also much loss.
The dry substance is then dissolved in pure water, 10 g. in
the litre, and after complete disintegration by agitation in a
stoppered bottle, is allowed to deposit less soluble salts and im-
purities, decanted or siphoned from these, and then filtered by
2:>ressure as described.
The filtered solution is now treated with enough hydrochloric
acid to effect complete precipitation of all kephalin as hydro-
chlorate, a salt which appears in voluminous flakes, and on
shaking collects on the surface of the liquid. (This precipitation,
like others to be related, affords a good criterion of the previously
dissolved state of the substance.) The mother-liquor is drawn
from underneath the precipitate with a siphon ; fresh water is
poured on the precipitate, and the whole is now placed upon a
paper filter or cloth, and washed Avith water until it begins to swell
and dissolve. At that point it is found that all HCl is washed out,
and that only pure liydrated keijlialin remains on the filter. This
was expressly proved by analysis in several cases, but more
especially in the following experiment.
Expulsion of Eydrochloric Acid from Kephalin by Water. — Half a
litre of a weak kephalin solution was precipitated by HCl and
filtered ; the precipitate on filter was placed in 500 cc. of water to
test its solubility, and was found insoluble, but the water con-
tained free HCl. It was again filtered, and after the solution had
run through absolute alcohol was blown on the precipitate ; this
became adhesive, and the solution refused to filter. The alcohol
and precipitate were transferred to a beaker, and the whole was
gently warmed in a water-bath to a temperature not much above
60°. The precipitate fused, but dissolved very sparingly,
apparently the less the more the alcohol made the precipitate
anhydrous. The alcoholic solution was now filtered off warm,
and the undissolved portion of the precipitate taken up with
ether. The solution so obtained was of a colour resembling
ordinary kephahn in ether, and was poured into the alcoholic
solution. The precipitate which ensued was fawn-coloured,
adhesive, and leathery ; it was isolated, washed with absolute
54 CHEMICAL CONSTITUTION OF THE BBAIN
alcohol, the mother-liquor being squeezed out of it, then pressed
and dried in air-pump. The whole was analysed for chlorine as
follows. It was heated to dryness in a strong solution of pure
soda, with carbonate and nitre added, and the mass was burned.
The fused mass was dissolved in dilute nitric acid, and the solu-
tion tested with silver nitrate. No ^precipitate occurred,
showing that ili e body ohtained in this process is not a hydrochlovate,
but kephalin in a j^urified state.
Bases and Salts wJdch are in combination tvith Kephcdin after Filtra-
tion of its Aqueous Solution. — The solution of hydrochloric acid and
other matters filtered from the precipitated kephalin was evaporated
to dryness. A portion was then boiled with solution of barita,
when traces of ammonia were evolved. The rest of the residue
was then ignited in a platinum dish to destroy all traces of
organic matter. The ash was slightly molten, and only partially
soluble in water, but easily soluble in water slightly acidified with
hydrochloric acid. The solution was filtered from a trace of
carbon, and treated with excess of ammonia, whereupon an abun-
dant i)recipitate of earthy salts fell down, and the solution assumed
a deep blue colour. Precipitate and solution were separated l)y filtra-
tion. The precipitate dissolved readily in a little HCl, forming
a slightly red solution, indicating presence of iron, which was con-
firmed by the sulphocyanide test. In another portion of the
solution dilute sulphuric acid gave an abundant precipitate of
gypsum, showing presence of ccdciuui. In another portion the
molybdate test showed the presence of p'^^osphoric acid. To the
remaining portion a few drops of ferric chloride were added, then
sodic carbonate nearly to neutrality, and lastly, excess of baritic
carbonate. This mixture was allowed to stand, filtered, and the
filtrate, freed from excess of barita by sulphuric acid, was again
filtered. The filtrate, after supersaturation with ammonia, gave
a great precipitate with ammonium oxalate, insoluble in acetic
acid, showing presence of much calcium. The filtrate from this
calcic oxalate on concentration and treatment with ammonia,
ammonium chloride, and sodium phosphate, gave the j^recii^itate
characteristic of magnesium. The precipitate produced by barium
carbonate was boiled with excess of pure soda, and the filtrate
warmed with ammonium chloride, when only a turbidity was
produced, indicating the absence of aluminium.
The alkaline filtrate from the foregoing precipitate by ammonia
PHOSPHOPJSED PPJNCIPLES OR PHOSPHATIDES. 55
was tested for lime by oxalate, when a considerable precipitate
was produced, showing the presence of lime tmcombined with jjJws-
pJioric acid, and which must therefore have been in combination
with part of the kephalin. The blue solution was again filtered
from calcium oxalate, which had been entirely precipitated, and
acidified with hydrochloric acid. The copper was precipitated by
hydro thion, the filtrate evaporated to dryness, ignited, and the
residue tested for alkalies. This residue was considerable, sur-
mounting in quantity or bulk the bases previously removed. It
was fusible with ease, and on solidification became white and
crystalline, but interspersed with many red particles of ferric
oxide. The fusion showed it to be mainly potassium chloride, but
there was also some sodium chloride present, as indicated by the
flame reaction, and the ferric oxide which had escaped precipita-
tion by excess of ammonia.
It is therefore proved that the kephalin obtained by the alcohol
and ether processes, and purification by solution in water and
filtration, consists of kephalin in the free state ; and of kephalin
combined with ammonium, sodium, potassium, calcium, iron,
copper, and with calcium and magnesium phosphates.
This experience was repeated a great number of times on, in
the aggregate, several hundred grammes of dry kephalin ; the
calcium and potassium salts were always found prevailing greatly
in quantity over the others ; none were ever absent. Although
I have not compared the total of the neutralising power of the
bases with the total of the acid-combining power of the kephalin,
by direct quantitative experiment, I am sure that much kephalin
must have been present in the free state, as will appear from
future developments concerning the combining and dissociating
powers of this body.
Dialysis of Kephalin. — A solution of 5 g. of crude kephalin v/as
dissolved in 500 cc. of water, and formed a white thick liquid,
from which some cholesterin crystallised on standing. It was
then placed on a dialyser of parchment paper. A trace of
kephalin passed into the water, but so small was the quantity
that no chemical operation could be undertaken with it.
Kephalin does, practically, not dialyse, but acts as a colloid,
and allows its impurities to pass out into the water.
Clarification and Decolonisation of JFaterij and Ethereal Solutions
of Kephalin. — To a solution of 1 g. kephalin in 100 cc. of water,
56 CHEMICAL CONSTITUTION OF THE BRAIN.
5 cc. of filtered fresh ivhHe of egg were added, and the mixture
was heated in a flask in the water-bath ; it remained turbid, and
no separation of coagulated albumin took place. The mixture
had a faintly alkaline reaction. On addition of a drop of acetic
acid to the heated mixture a copious precipitate ensued, which
enclosed both kephalin and albumen (the kephalin solution b}'
itself, cold, is only partially or imperfectly precipitated by the
same acetic acid). By filtration a perfectly clear liquid was
obtained, which was no longer precipitated by barita water or
platinic chloride, and not changed by boiling. Consequently, all
albumen and all kephalin were removed together from the solu-
tion, and perhaps in part combined. Cold absolute alcohol in
large quantity extracted all or nearly all kephalin from the albu-
men, and on distillation left it ijerfedly u-Mte. The albumen, on
the other hand, was, after washing with alcohol and ether, in a
finely divided pulverulent state, and not hard nor horny. This
process is therefore useful for preparing snow-white kephalin,
which must, however, not again be brought into contact with
ether, as that would immediately cause it to become coloured
under the influence of oxidation,
Ivfiuence of Animal Charcoal on Water Solution of Kejjhalin. —
To a solution of 1 g. kephalin in 100 cc. of water, 2 g. of pure
animal charcoal were added, the mixture shaken, and then sub-
jected to the vacuum filter. A little fluid passed, which became
at last quite clear. A portion of the last clearest, collected by
itself and tested, was found to be almost pure water ; for hydro-
chloric acid, platinic chloride, barita hydrate, and lead acetate,
l^roduced the very feeblest precipitates only, while the original
solution was made solid by the same precipitants. The animal
charcoal therefore retained the kephalin, and when isolated and
extracted with alcohol yielded it up to that solvent. This ex-
perience, as well as that made with albumen, shows that the
watery solution of kephalin cannot be clarified by these agents, if
indeed they do not show also that the condition of kephalin is
one of suspension and not of true solution. That, however,
charcoal has a special attraction for kej^halin, such as it also
exhibits towards other ammonium ])ases and alkaloids of un-
doubted solubility in water, is shown by the following experiment.
Bearing of Kephalin in Ether tcith Charcoal— A concentrated
solution of kephalin in ether was treated with much animal char-
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 57
coal, in order to be decolorised. The object was but partially
obtained. The charcoal, after filtration and washing, was found
to retain much kephalin, which was extracted by boiling absolute
alcohol, and from this deposited on cooling in a perfectly white
state. The solution deposited more on spontaneous evaporation.
Both were tested and identified as kephalin.
Kephalin therefore can be removed from watery solution by
charcoal and curdling albumen, and again extracted from these
substances by hot or cold alcohol in large quantity, and obtained
from these solutions in a perfectly white state.
Ultimate Analysis of Kej^halin. — A specimen of highly purified
kephalin was passed through the water-filtration and hydrochloric
acid process ; it amounted to four litres of one per cent, solution,
and after resolution in ether and precipitation by alcohol, left
about 30 g. dry matter. It was thoroughly dried in vacuo over
sulphuric acid, being frequently triturated, and ultimately reduced
to a fine powder. Carbon and hydrogen were determined by
combustion with lead chromate and copper turnings. Nitrogen
was determined by volume, the bichromate and carbonate mixture
being used for production of carbonic acid gas. Phosphorus was
determined by evaporating the substance with solution of pure
soda, made from metallic sodium, mixed with carbonate and
nitre, to dryness, slowly deflagrating, etc., and determining phos-
phoric acid by magnesia method.
Thus were obtained the following percentages and atoms :
Theory.
Eound.
~ by At. Wgt.
~ by N = l
Atoms.
Per cents.
C 60-00
5-00
41-7
42 C
504
60-28
H 9-39
9-39
78-3
79 H
79
9-44
N 1-68
•12
1-0
N
14
1-67
P 4-27
•13
1-0
P
31
3-70
0 24-66
1-54
12-9
13 0
208
24-88
100-00
leading to formula C^^H.^NPO^^ = C^.^H^gNPOg + 5Bfi.
We shall see hereafter that this formula is supported by the
results of the analysis of a number of other preparations, being
partial or complete compounds of kephalin, of which the organic
matter always has the composition of the free substance ; the
results of a series of chemolytic experiments lead to the latter
formula. But the assumption of the presence of five molecles of
58 CHEMICAL CONSTITUTION OF THE BRAIN.
water of hydration brings the constituent hydrogen to so low a
figure that it cannot be explained out of the sum of the chemolytic
products. On the other hand, the assumption of the presence of
five atoms of loosely attached oxygen meets also with theoretical
objections. The difficulty here touched requires evidently ex-
perimental elucidation.
SolubUUIes of Kephalui. — In water kephalin swells and forms an
emulsion, ultimately an imperfect turbid solution. Its affinity
for water is very great, and the List quantity of water is expelled
from it in the vacuum only with great difficulty and after a long
time. From a watery solution or mixture it cannot be extracted
by ether, as the liquids form an emulsion which persists even after
a portion of the ether has separated from the water. This emul-
sion is very thick, like paste, white like milky water, and practi-
cally unmanageable. When a drop or a few drops of a concentrated
ether solution are allowed to fall into a test-tube full of water, the
mixture is at once transformed into a white jelly, which is so firm
that the tube can be turned upside down without anything flowing-
out of it. Solutions of kephalin in water on standing do nob
decompose or become mouldy, even in the course of some weeks.
Cold absolute alcohol dissolves a little kephalin, more on boiling,
and deposits a part on cooling in white flocks. One hundred
parts absolute alcohol at 17° C. dissolve seven parts ke^^halin ; at
boiling heat of the alcohol nine parts, of which two parts are
deposited on cooling. AVhen an excess of kephalin is boiled with
an insufficient amount of alcohol, the j^art which remains insolul)le
does not seem to undergo any change, for it retains its solubility
in ether and precipitability by alcohol and other reactions.
In ether kephalin, when not too much hydrated, is highly
soluble ; when dry it is soluble in anhydrous ether in almost any
proportions ; it does not crystallise from this solution, and cannot
be made to deposit as from a mother-liquor. It is precipitated
from the ether solution by an equal or greater volume of alcohol
in white clouds, which combine to clots, and ultimately forms a
firm substance, which becomes at first plastic, and then dries in
vacuo to a hard brittle mass. The ether solution becomes quickly
red in transmitted light, and fluoresces with a fine green colour.
No other phosphorised or other brain ingredient shows this pecu-
liarity except kephaloidin.
A specimen, tln-icc precipitated from ether by alcohol, and
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES, 59
when last in ether, exposed during twenty-four hours in ice to a
temperature of 0°, proved soluble in cold benzol, and very soluble
in hot. It formed a yellow solution. A sample in a test-tube
exposed to frost gave no deposit. The addition of alcohol to the
benzol solution produced a slight precipitate, insoluble in excess
of alcohol, but soluble in excess of benzol, and soluble on heating.
Benzol can therefore not be used, like ether, for the purification
of kephalin.
Reactions of the Aqueous Solution of Keijhalin. — 1 per cent,
solution, filtered by air-pressure through three-fold Swedish filter-
paper, was used.
1. Hydrochloric acid gives a bulky curdy precipitate, slightly
yellow, and after isolation soluble in ether, not precipi-
tated b}^ alcohol from its ethereal solution. (In the
filtrate from this hydrochloric acid precipitate platinic
chloride produces the merest opacity.) But from the
ethereal solution of the HCl precipitate alcoholic PtCl_,^
throws down a precipitate, which is soluble in ether,
and reprecipitated by alcohol.
2. Sulphuric acid produces a precipitate like that produced
by HCL
3. Nitric acid the same as the previous acids.
4. Barita water produces a bulky curdy precipitate.
5. Lime-water produces a similar precipitate, but it does not
separate like the BaH^O^ precipitate.
6. Cadmic chloride induces a curdy precipitate which readily
coalesces.
7. Zinc chloride behaves similar to CdCl.,.
8. Mercuric nitrate produces a dense precipitate, which is in-
soluble in nitric acid, but coloured slightly yellow
thereby, heat being evolved. The precipitate is some-
times rose-red, and in adhesive flakes. Washed and
allowed to stand with water it becomes again white,
ropy, and adhesive, and soft, and on being shaken easily
dissolves in water in the manner of the original kepha-
lin. The mercuric nitrate seems therefore to be separated
by water from kephalin in the same manner as other
salts and acids are.
9. Barium chloride produces a good dense flaky precipitate.
Immediately after isolation it is insoluble in water, in-
soluble in alcohol, but easily soluble in ether, and
apparently reprecipitated by alcohol. This reprecipitated
matter contains barium, but gives it up again to water.
10. Calcium chloride acts like BaCl.^.
11. Platinic chloride produces a bulky precipitate.
60 CHEMICAL CONSTITUTION OF THE BRAIN.
12. Ammonia makes solution a little turbid, but causes no pre-
cipitate.
13. Platinic chloride mixed with HCl produces a very well-
defined precipitate.
14. Magnesium chloride causes a very precise immediate preci-
pitate.
15. Ferric chloride produces a yellowish turbidity and imperfect
precipitate.
16. Uranic nitrate produces a white turbidity and imperfect
precijjitate.
1 7. Watery bromine produces a bulky, nearly white precipitate,
soluble in caustic potash ; acetic acid added to this again
liberates the precipitate. Chloroform added to this mix-
ture produces a chloroform solution of Br at the bottom,
containing the excess of reagent, and an impracticable
white emulsion on the top.
18. Cupric nitrate "J
19. Cupric chloride f All produce perfect precipitates of a
20. Cupric sulphate ( greenish-white colour.
21. Cupric acetate )
22. Mercuric chloride makes the solution very turbid, but pro-
duces no precipitate.
23. Mercuric acetate causes an immediate complete precipitate.
24. Silver nitrate, immediate complete precipitate, darkening a
little when exposed to sunlight.
25. Gold terchloride, and a drop of hydrochloric acid, cause
an immediate precipitate, which blackens over night.
26. Antimonic chloride produces a very bulky precise white
precipitate.
27. Stannous chloride, a white flaky complete precipitate.
28. Stannic chloride, a precipitate and turbid solution.
29. Tannin in water, no particular reaction.
30. Picric acid, a turbidity, but no manageable precipitate.
31. Arsenious acid, a precipitate and turbidity.
32. Arsenic acid, a very complete immediate precipitate.
33. Phosphoric acid, a very complete immediate precipitate.
34. Basic lead acetate, a precipitate and very turbid solution ;
no perfect separation.
In none of the foregoing reactions was any artificial heat em-
ployed, but they were all made at the ordinary temperature.
It was found that most of these precipitates could not be
washed with water without losing either acid or base, or salt,
with which they were combined. But most of them remained in-
soluble in water until the point of i)urity was reached, when the
kephalin either dissolved in the pure water, or clogged the
filtering-paper.
mOSPHOPdSED PRINCIPLES OR PHOSPHATIDES. 61
Compounds of Kephalin,
Kephalin Cadmium Chloride. — Two litres of a filtered 1 per cent,
solution were precipitated by dilute HCl. The mother-liquor was
drawn off, and the precipitate washed by agitation with water.
The washing-water was again drawn off, and watery solution of
cadmium chloride added, which caused great condensation of the
precipitate. The liquor was again drawn off, and the precipitate
shaken violently with a great quantity of alcohol containing
alcoholic cadmium chloride. Thus the precipitate was condensed
to a viscous mass, from which the mother-liquor was drawn off,
and the alcohol entirely removed by careful manipulation. After
draining, the precipitate was dissolved in ether, and to the ethereal
solution was added alcoholic cadmium chloride cautiously, till a
slight permanent precipitate was perceived. This was removed
by filtration, and the brilliant fluorescent filtrate precipitated by
absolute alcohol. The viscous mass was again drained from all
alcohol by pressure with a glass rod. Kedissolved in ether, it
formed a perfectly clear solution, which was reprecijntated by
absolute alcohol, when the compound came down in an almost
pulverulent state. It was thrown on a filter, washed with absolute
alcohol, then removed on a glass dish and placed under a dryer,
then in a vacuum over sulphuric acid, and frequentl}^ removed to
be powdered in a mortar, and ultimately finely pulverised. It
was then subjected to elementary analysis. Carbon and hydrogen
were determined by combustion with PbCrO^, and copper turnings.
Nitrogen was determined as gas. Chlorine, cadmium, and phos-
phorus were determined by fusion with caustic soda, nitre, and
carbonate, solution of salts in acid, precipitation of cadmium by
hydrothion, and conversion into carbonate. Chlorine and phos-
phorus were determined in filtrate from cadmic sulphide by the
usual methods. The cadmium and chlorine were in the relation
of Cd : CI2, inasmuch as 4*15 parts chlorine require 6-54: parts
cadmium.
Summary of per cent, found :
C
H
P
O
53-603 ]
8-519 i
1-37 \ 89-38
3-54: I
22-35 J
Cd
CI
10-62
100-00
100-UO
62 CHEMICAL CONSTITUTION OF THE BRAIN
Calculation shows that the kephalin is not completely saturated
with CdCl2; that about four parts out of nine are uncombined.
For the formula, derived from the organic matter with P as 1,
i.e., Qj^^^^V0y^,Q^Q\.2 yields the equation —
C42H^c,NPOi3,CdCl2 CdCl,
1019 : 183 - 100 : 17-95,
whereas only 10*62 per cent. CdCl^ were found.
Deducting CdCU, and calculating per cents, of elements in
organic body, we get —
-r- by At. Wgts.
-^ by P as 1.
At. Wgts
c
59-97
4-99
41-6
504
H
9-53
9-53
79-4
79
N
1-.53
•11
1-0
14
P
3-96
■12
1-0
31
0
25-00
1-56
13-0
208
836
leading to formula C^gH^gNPO^g, with an atomic weight of 836,
thus fully sustaining the composition of the free substance.
As 10 parts of CdCl^, supposed to be combined with a molecle
of kephalin, correspond to 59 parts of compound, about 40 j^er
cent, of the above substance may have been free kephalin.
The very weak chemical affinities of kephalin are here exhibited
in a striking manner ; CdCl2 was brought into contact with it at
various periods, and yet could not be retained in combination.
This is due to the circumstance that CdCl2 cannot be dissolved in
absolute, but only in somewhat watery alcohol ; and even little
water decomposes the compound, and carries the CdCU away, as
will be shown by special experiment hereafter.
Kephalin u'ith Hydrochloric Acid and Flatinic Chloride. — About
three litres of 1 per cent, solution, which by the deposition of the
insoluble part and filtration had lost much of the 1 per cent,
originally dissolved, were treated with a mixture of HCl and
PtCl^ in slight excess. A bulky yellowish-white curdy precipitate
ensued, and rose to the surface ; it was allowed to contract, and
the yellow mother-liquor drawn from beneath it by a siphon.
Absolute alcohol, equal in bulk to the precipitate, was now 2)0ured
ui)on it, and the mixture violently shaken. This caused a further
contraction of the precipitate, indicated by lesser bulk, increase of
the yellow colour, and production of adhesiveness. The alcoholic
PIIOSPHORISED PRINCIPLES OR PHOSPHATIDES. 63
liquid was again siphoned off, and another quantity of absohite
alcohol was now poured on the precipitate, and the mixture
violently shaken. The precipitate thereby became deep yellow,
adhering to the glass, and so contracted that the liquor could be
poured off quite clear. The precipitate was now treated with a
minimum of ether, in which it proved quickly and entirely soluble.
On filtration, nothing whatever remained on the filter. An equal
volume of absolute alcohol was added to the ether solution,
whereby almost the whole of the salt was precipitated. The
latter was freed from mother-liquor by careful manipulation with
a glass rod, redissolved in absolute ether, which gave a brilliant
solution requiring no filtration, and this was reprecipitated by
absolute alcohol in equal volume, added in a thin stream while
the liquid was being stirred. The mother-liquor was poured off ;
the precipitate, which immediately became brittle and hard on
contact with absolute alcohol, was drained from alcohol and placed
over sulphuric acid in the vacuum.
Another amount of the same body was made from four litres
of a 1 per cent, solution in the same way, with this difference —
that whereas in the first case the precipitate was washed with
alcohol directly, in this case two washings with water were carried
out before the application of alcohol. This was done in order to
test the effect of water upon the compound desired to be pro-
duced. It was found that the dry body treated with ether and
alcohol like the first preparation contained only a trace of platinum
and a '/estige of chlorine, both elements being too small in
quantity for accurate determination.
Both preparations were therefore united, and it was sought to
combine them with platinic chloride under circumstances where
the influence of water was as much as possible excluded. (It
must be remembered that solid platinic chloride contains six
molecles of hydration water, which it necessarily carries into all
its solutions.) They were dissolved in ether, and an ethereal
solution of PtCl^ was added, then precipitated with absolute
alcohol ; again dissolved in ether, and treatment with PtCl^ re-
peated. Ultimately the precipitate was dissolved in pure ether,
reprecipitated by pure alcohol, and dried in vacuo.
Analysis gave 3-592 per cent. Pt, and 3-265 per cent. CI. If
the platinum had been accompanied by only as much chlorine as
corresponds to tetrachloride, 2-58 per cent. CI should have been
64 CHEMICAL CONSTITUTION OF THE BRAIN
found. By calculation we find that there is one-fifth of CI more
than corresj^onds to this proportion, viz., 1 Pt: 5 01 = 3-592 Pt :
3-22 CI.
A compound of the presumable formula :
2(C^^H^c,NPOi3) + 2ClH + PtCl4 with an atomic weight of 2083
requires 9-5 per cent. Pt and 10-2 CI. Consequently, the platinic
chloride compound comprises about one-third of the kephalin, of
which two-thirds are uncombined.
It is thus seen that although kephalin is most completely pre-
cipitated by PtCl^ from its watery solution, yet by the process of
solution and precipitation with solvents in which PtCl^ is soluble
most of the PtCl4 is extracted from the combination and lost in
the mother-liquors, just as it is almost entirely extracted from the
same precipitate by water. In short, the acids, bases, and metallic
salts which easily combine with kephalin when they are present
in excess, are rapidly separated from it by solvents in which they
themselves are readily soluble.
Chemically speaking, these results are disappointingly negative,
inasmuch as they refuse to furnish the ordinary means for deter-
mining the atomic weight and for finding guarantees of purity of
preparations. But physiologically these features are of the
greatest interest, inasmuch as they show us a marvellous diversity
of power of reaction of kephalin, by its entering into and out of
combination according to external circumstances. When the com-
binants are off"ered in a concentrated state they are retained ;
when the liquids which carry the combinants (blood, serum,
cerebrospinal fluid) become again diluted, the combined matters
must again pass into solution and travel further. Thus every
change of chemical composition of the juices of the body must
necessarily and powerfully affect the condition of the brain
and nerves, and of all tissues and cells containing their specific
ingredients.
h. Amldohephalin.
The details of the observation regarding this principle, and of
its transformation into lead salt, Avill be given lower down, in the
section of the dinitrogenised monophosphatides.
c. Oxilcephalin with Cadmium Chloride^ C42H^c)NPOi4,CdCl2.
When the white matter (Ox) has been extracted with ether and
the kephalin removed from the ether solution by precipitation with
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 65
absolute alcohol, there remains a bulky solution containing all
lecithin, much sphingomyelin, and some kephalin, together with
the cholesterin previously contained in the white matter. When
to this solution CdCl2 is added, a voluminous precipitate ensues,
which, after washing, yields to ether a quantity of coloured salt.
This after concentration is precipitated by alcohol, and purified
by repetition of this treatment. The composition of this precipi-
tate is shown in the following summary of analyses :
In 100 of
Per cents, found. -r- by At. Wts. -^Cd as 1. organic matter.
C 48-12 ] 4-01 42-21 58'71
H 7-55 \ 7-55 79-47 9-21
N 1-43 \ 81-95 0-102 1-07 1-74
P 3-524 I 0-113 1-18 4-30
O 21-33 J 1-33 14-00 26-02
Cd 10-65 \ 0-095 1-00 99-98
CI 7-40 / ^ 0-208 2-18
100004 100-00
leading to formula C^gH^gNPOj^, CdCl2.
This compound is noteworthy on account of two features, viz.,
that it coincides with the composition of the theoretical CdCl^
salt of kephalin, plus one atom of oxigen, and that the CdCl^ is a
complete molecle, combined with an ap^^arently complete molecle
of organic matter. Such compounds as this and the ones to be
described hereafter with 15 atoms of oxigen, make one regret
that there are no means of determining oxigen directly in organic
chemistry. The oxigen is estimated by the void left by the sub-
stances determined, and this gives an opportunity for small im-
purities to be summed up under the guise of this element. Now
the substance here considered had not undergone the process of
purification by water, filtration, and acid, and it may therefore
have been kephalin to which some slight impurity was attached.
On the other hand, there is no proof of the existence of such im-
purity, and none could be found by testing. It is therefore
necessary to consider this substance as a genuine compound of a
kephalin containing an atom of 0 more than the normal kephalin,
to which it will be convenient to apply the term oxikephalin. In
any case the isolation of this body from the mother-liquor of
kephalin by the ether process, by means of CdClg is of sufficient
5
66 CHEMICAL CONSTITUTION OF THE BRAIN
importance in itself, no matter how the question of the atom of
oxigen may ultimately be decided by further research.
Behaviour of a similai- Salt with Water. — A portion of a salt
similarly obtained, though not analysed, was digested with w^Jer,
whereupon it began to swell, and the water after filtration was
found to contain large quantities of CdCU, proved expressly by
the hydrothion and silver tests. When the extraction with water
had been continued for some time filtration was effected, when
during washing the body swelled to such an extent as to clog the
filter. There was therefore no guarantee of its purity from CdCl^.
The experiment proves that by simple digestion with water much
CdClg is extracted, but the entire amount can only be removed by
a \oT\g process of dialysis in corrugated filters of vegetable parch-
ment. The compound cannot be freed from cadmium by H^S, as
when so treated in ether solution it only assumes a yellow colour,
and the CdS remains dissolved. This peculiar bearing is ob-
served by several phosphorised compounds.
d. Peroxikephalin, C^^yj^VOy^.
A quantity of kephalin, obtained after frosting the ether solu-
tion and precipitating it by absolute alcohol, was subjected to
elementary analysis, without having undergone the water filtration
and HCl process, and gave results of which the following is a
summary :
In 100. ^ by At. Wts. -r- by N = 1.
C 57-750 4-8125 42-85
H 8-902 8-9020 79-26
N 1-573 0 1123 1-00
P 3-680 0 1187 1-05
0 28-095 1-7560 15 63
leading to formula C^.^H^gNPOi^ ; at. w. = 868.
Transformation of this Body into Lead Salt. — About 10 g. of the
analysed substance were dissolved in ether, and to this solution a
warm alcoholic sohdion of lead acetate was added. A viscous pre-
cipitate was produced, which settled in a mass. The lead acetate
was not used in excess, the addition being discontinued while
there was still a little matter in solution admitting of precipita-
tion. The mother-liquor was poured off, the mass of the precipi-
tate was stirred and rinsed with a little ether first, and afterwards
with absolute alcohol. The precipitate was now digested with a
quantity of ether, which dissolved much and left a portion un-
PHOSPHORISED RRINCIPLES OR PHOSRHATIDES. 67
dissolved, which was disregarded. The solution was filtered off
and precipitated by absolute alcohol, the precipitate was washed
four or five times with absolute alcohol, dried and analysed.
The compound dried at 80° C. was fused with soda, nitre, and
carbonate mixture, the lead precipitated by H2S, and the PbS
transformed into PbSO^ by ignition with HNO3 and H^SO^.
Summary :
Per cent, found requires
C 38-337 39-436
H 5-760 5-868
N 0-9755 1-095
P 2-717 2-425
0 20-312 18-782
Pb 31-869 32-394
100-000 100-000
It w411 be seen by a comparison of the oxigen quantities in the
free body, with those of the salt, that there is no reason for
assuming lead to be present as oxide ; on the contrary, the H
being less in the organic part of the lead salt than in the free body
justifies the assumption of a substitution of by Pb2.
Comparison of the Organic Matter in the Lead Salt, with the Com-
position of the Free Body and of the Organic Matter in a Salt of
Kephaloidin with CdCl^.
Per cent, found in original Per cent, found in CdCl, Per cent, found in lead
free peroxikephalin. salt of kephaloidin . salt of peroxikephalin.
C 57-75 57-91 56-31
H 8-90 8-82 8-30
N 1-57 1-67 1-43
P 3-68 3-71 3-98
O 28-09 27-78 29-81
The phosphorus, as in nearly all analyses, is found somewhat
too high, rising in the lead salt to 5 as compared with N as 4.
But on the whole the change by the removal of some insoluble salt
is not so great as to negative the assumption that the free body
and body contained in the lead salt have essentially the same com-
position, more particularly the proportion of oxigen has not been
decreased hij the comhination.
e. Kephaloidin.
Definitior\.. — The substance thus designated is much like kephalin
as above described and may be identical in composition with it,
5—2
68 CHEMICAL CONSTITUTION OF THE BRAIN,
but presents some slight differences, which necessitate a pre-
liminary distinction. It is obtained from buttery matter ; kephalin
from white matter. It is more fluid than kephalin when first
precipitated, and never dries to the same hard brittle substance
as kephalin, but presents a fused appearance. It presents the
same irregularities in its combinations as kephalin ; it farms
oxikephaloidin, which, like oxikephalin, combines with a molecle
of CdCl.,, forming a pretty concise salt.
Solubilitj/ in Water and Filtration. — Five g. of dry, hard, but
plastic kephaloidin were dissolved in 500 cc. of cold water with
trituration. The substance became mucous at first, and on agita-
tion in a long cylinder was disintegrated, and a turbid emulsion-
like solution resulted. This was passed through the pressure-filter,
and passed easily ten layers of English filtering-paper. Next ten
layers of Swedish paper were employed, when an entire atmosphere
of pressure allowed the liquid to pass, but slowly.
Bearing in Dialysmg Apparatus. — First Experiment.— The fore-
going solution, after filtration and subsequent treatment with
animal charcoal, which did not make it clear, was distributed over
two dialysers of parchment-paper. After twenty-four hours the
dialysate gave but slight evidence of containing kephaloidin, by
giving a mere vestige of precipitate with lead salt, while the
original solution gave a very copious precipitate. Only a very
minute portion of matter, therefore, had passed the diaphragm.
Second Experiment. — A solution and emulsion of about 20 g. of
kephaloidin in 500 cc. of water was, without having been filtered,
subjected to dialysis. After twenty-four hours only a very small
quantity of kephaloidin had j)assed, as shown by the lead precipi-
tate. The original solution on the dialyser gave a copious thick
precipitate with the same lead salt. It was thus shown that a
1 per cent, solution can hardly be dialysed, while a per cent,
solution dialyses a little, but not enough for practical purposes.
Kephalin and kephaloidin act in watery solution like colloids, and
remain on the dialyser, while allowing the crystalloids mixed or
combined with them to pass in the pure water. They act as
dialysers themselves when placed in pure water, and yield up
the soluble salts or bases or acids with which they are combined.
Dialysis by vegetable parchment is effective in completing this
process.
Bearing of the Ellier Solution icith Water. — The kephaloidin was
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 69
twice precipitated by alcohol from ether solution, and then redis-
solved in ether. A few drops falling in water are precipitated,
and on shaking a turbid emulsion is formed. Boiling transforms
this into a turbid mucous mass w^ith thick and viscid flakes.
Hydrochloric acid added to this emulsion causes white curdling,
and the white flakes can with difficulty be filtered off. The
filtrate is white and turbid.
Water, hydrochloric acid, and ether in certain proportions pro-
duce a thick white jelly; a little more ether separates oily ether
solution, which gives no precipitate with alcohol, or in this
mixture with CdCl^, and contains therefore HCl.
Ether solution mixed with little water becomes a solid white
mass; more water produces curds, ultimately emulsion and
solution.
Kephaloidin is easily soluble in benzol ; treated with HCl gas,
this solution changes colour, but gives no precipitate ; alcohol
added to this gives a little precipitate, soluble in excess ; ether
gives no precipitate in the benzol HCl solution. A solution of
kephaloidin in anhydrous ether is not precipitated by benzol.
Reactions of the Watery Solution of Kephaloidin :
Hydrochloric acid produces a dense slightly yellow precipitate,
Avhich after isolation is soluble in ether and not precipitated
by alcohol. In the ethereal solution of HCl precipitate,
alcoholic PtCl^ produces a precipitate soluble in ether and
reprecipitated by alcohol.
Sulphuric acid produces a precipitate like that by HCl.
Nitric acid, same as sulphuric.
Barita water ^
Lime water ( All produce good precipitates which coalesce
Cadmium chloride i' well on standing or agitation.
Zinc chloride j
Mercuric nitrate gives a good curdled precipitate insoluble in
HNO3, but made slightly yellow thereby.
Barium chloride also produces a good flaky precipitate. This
body after isolation is insoluble in water and absolute alcohol,
but readily soluble in ether.
Platinum chloride gives a complete precipitate.
Lead acetate gives a good precipitate.
Mercuric acetate gives a very voluminous precipitate.
Cupric acetate, a whitish flocculent precipitate.
Kephaloidin Lead. — A specimen of kephaloidin which had been
frozen in the ethereal solution, and precipitated by alcohol, was
70 . CHEMICAL CONSTITUTION OF THE BRAIN.
once more dissolved in ether, and placed in a freezing mixture
for twenty-four hours. No deposit occurred. This ether solution
also gave the reactions above described, and with silver nitrate its
watery solution gave a copious white precipitate. The ether
solution was poured slowly in a thin stream into absolute alcohol,
when the kephaloidin was precipitated as a viscid mass. The
whole of this was dissolved in water, and lead acetate added ; a
copious precipitate ensued, which was filtered and washed, ex-
tracted with warm dilute alcohol, ultimately with warm absolute
alcohol ; it then dissolved in ether without residue, was precipitated
by absolute alcohol, became pulverulent, was dried in vacuo, and
analysed.
Summary. matS"" Theory of C,,H,,NPO,,
C 50-983 1 60-88 60-28
H 7-721 I rp , 1 9-22 9-44
1 A1- I iotal organic . r,-
N 1-01, >. go - « 1-21 1-6/
P 3-666 I ^'^ 4-37 3-70
O 20-353 J 24-32 24-885
Pb 16-260 16-260
100-00 100-000
100-000 100-000
It is at once evident that the Pb stands in no simple proportion
to any other element. Kephalin lead if dibasic would require
19 per cent., if monobasic 11 per cent., of Pb. The molecle of
the kephaloidinate contains thus rather more than half a molecle
of Pb, and is consequently a mixture of lead salt with free body.
There is also an irrationality perceptible on the P, which is too
high, and the N, which is too low. But on the whole the consti-
tution, properties, and products coincide with those show^n by
kephalin of the compared formula.
/. Oxikej^haloidin ivifh Cadmic Chloride, 2(C^.H-.NPOi4) + CdCl,.
In this case a quantity of kephaloidin obtained by the ether
process, not purified by water, filtration, and HCl, etc., was trans-
formed into CdCl, salt, and precipitated by alcohol ; redissolved
in ether, and the solution repeatedly frozen, and freed from some
deposit, then precipitated by absolute alcohol. It could be dried
at 80° C. without change.
PHOSPBORISED PRINCIPLES OR PHOSPHATIDES. 71
Summary of analyses per cent, :
C 52-796 V
H
P
0
Cd
CI
? Ia^ I Total organic
3-690. ^^^^^
25-032
5-410
3-430
100-000
-840
100-000
leading to formula C^gH^^NPOi^ for organic matter. If one atom
of CdCl2 were combined with two atoms of organic body, then
9 -8 per cent. CdCl2 should have been present.
Comparison of the Composition of the Organic Matter with
Per cents, of
Organic matter of
oxikephalin with
CdCl...
C 58-71
H 9-21
N 1-74
P 4-30
0 26-02
Organic matter in
oxikephaloidin
with CdCIa-
57-91
8-82
1-64
4-04
. 27-45
Free per-
oxikephalin.
57-750
8-902
1-573
3-680
28-095
Organic matter in
lead salt of
peroxikephalin.
56-31
8-30
1-43
3-98
29-81
It will thus be perceived that the oxikephaloidin is inter-
mediate between oxikephalin and peroxikephalin in composition
in all items except alone hydrogen. This peculiar anomaly, if
such it be, must be reserved for future deliberation. The
empirical formula expressing the composition of this salt is
2(C,,H„NP0i,) + CdCl,.
The ethereal solution of this salt was not precipitated by
hydrothion gas passed through it.
. ' g. Decompositions of Kephalin.
When pure kephalin in the perfectly dry state is heated in a
water-oven to between 90° and 100° it fuses to a dark red
transparent viscid oil. It becomes solid again on cooling, but
retains a viscosity, so as to adhere to the fingers, which it did not
before it was heated. Treated with water, the heated and cooled
kephalin swells again and gradually dissolves, but is darker
72 CHEMICAL CONSTITUTION OF THE BRAIN,
coloured, so that some slight degree of decomposition or of oxida-
tion seems to have taken place.
When heated to higher temperatures it gives out much heavy
strongly smelling inflammable vapour, which partially burns, with
formation of much soot. Ultimately a bulky charcoal is left,
which cannot by ordinary heating in platinum vessels be entirely
destroyed, as it is soaked with phosphoric acid. It cannot be
entirely burned, even after the principal quantity of phosphoric
acid has been extracted with water. Complete combustion is
effected only in the presence of nitre.
With concentrated sulphuric acid dry kephalin in fine particles
immediately assumes a dark red-brown colour, which gradually
becomes nearly black. When sugar and sulphuric acid are allowed
to act upon kephalin, a reaction similar to the one given by bile-
acids is gradually engendered. But the process essentially requires
time, during which the mixture passes through a stage of dark-
brown colour, until a deep purple is at last attained. But this
colour is never so pure or beautiful as that obtained with cere-
brosides, myelin, or the bile-acids. It is therefore probable that a
decomposition of the kephalin has to be effected before the reaction
is attained.
A. Chemohjses of KejyhaUns.
a. Limited Chemolysis hij Caustic Soda. — A watery solution con-
taining 30 g. of pure kephalin, filtered, etc., was precipitated by
HCl. The pulpy deposit was put into a flask, and a solution of
5 g. crystallised soda hydrate added. The precipitate disappeared
on agitation, forming first a gelatinous, later a fluid solution. The
.solution was now placed in a bath, and heated gentl}^ for nine
hours. During the process it was observed that skins formed on
the surface similar to membranes on milk while being heated. On
cooling, the liquid formed a gelatinous cake, consisting of viscous
curds set in a thinner fluid. Next day the mixture was boiled
during nine hours on a sand-bath. Bumping was mitigated by
dropping a spiral of platinum into the fluid. Great frothing
ensued, which was opi)osed by a funnel fixed upon the top of the
flask by a cork, and supported by a stand. After this boiling the
liquid was still tur])id, became gelatinous, viscid, and set on
cooling, and covered by a membrane. On commotion it showed
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 73
the wavy glistening appearance of soaps, and when at repose in a
beaker formed folds as from a tubular membrane sinking.
Reactions of the Soapy Solution. — It was insoluble in water, and
not further precipitated thereby. It was curdled by cold alcohol,
dissolved by hot, leaving, however, some particles undissolved,
which proved soluble in ether. It was not precipitated by concen-
trated sodium chloride solution — i.e., not salted out.
Decomposition of the Soaps hy Hydrochloric Acid. — The soap was
now treated with hydrochloric acid, until a strongly acid reaction
was attained, when a whitish-yellow precipitate fell, and the fluid
lost its viscosity. The precipitate was washed with water, and the
filtrate was evaporated on water-bath.
The Precipitate of Fatty Acids on the Filter was washed, and be-
came very adhesive, yet fatty ; boiled in water it did not fuse,
but agglutinised a little ; nothing like oil or fused fatty acid
appeared. Warmed on paper it did not fuse like fat, and gave
only a very slight grease-stain ; most of it remained slightly
glistening on the surface, even when heated until brown. The
matter had a fine, smooth, greasy touch between the fingers, and
seemed like a fatty acid in a hydrated, swelled state. It dissolved
easily in cold absolute alcohol, leaving a quantity of adhesive dark
matter undissolved. The latter will not be considered any further
in this place.
The Acids soluble in Alcohol — Kephalophosphoric, Kephalic, and a
Third Acid. — The solution showed a feeble green fluorescence; it
was filtered from a slight secondary deposit of insoluble acid.
Alcoholic acetate of lead w^as now added, w^hich produced a bulky
nearly white precipitate. This was washed with absolute alcohol,
drained on paper, dried, and treated with ether. It was entirely
insoluble in ether, and constitutes
Kephalophosphate of Lead, so named from being the lead salt of
a phosphorised acid obtained from kephalin, more complicated
than glycerophosphoric acid. The total quantity of salt obtained
weighed 6-2 g. Powdered and dried at 95^ C, it baked together
a little on surface, but when stirred remained pulverulent.
A combustion for nitrogen gave only a trace of permanent gas,
so that the substance is proved to be free from nitrogen.
Phosphorus calculated from the Pb pyrophosphate found
-3-572 per cent. P.
74 . CHEMICAL CONSTITUTION OF THE BRAIN
Found in 100. -h by At. Wgts. ^ by P = 1
C 48-2fi2 4-022 35-3
H 7-990 7-990 70-1
Pb 23-840 0 1 151 1-01
P 3-572 0 1139 1-00
0 16-336 1-021 8-96
100-000
leading to formula Cy^HwQPbPOc,.
Deducting the lead, and calculating the 76-16 per cent, of organic
matter as 100, we get — •
-^ by At. Wgts. H-by P as 1. At. Wgts.
C 63-369 5-2807 34-9 420
H 10-491 10-4910 69-3 + 2 72
P 4-691 0-1512 1-0 31
O 21-449 1-3405 8-8 144
100-000 667
The atomic weight derived from the Pb pyrophosphate is 663,
which closely approximates the quantity found directly.
The conclusion drawn from this experience was that chemolysis,
with an amount of alkali insufficient to satisfy at least three
molecles of acid, developed from one molecle of kephalin, must
stop short of complete decomposition, and produce intermediate
products, of which the principal one is kephalophosphoric acid.
It is probably an acid containing three acid radicles and an alcohol
radicle —
Kephalyl )
Stearyl - Phosphoryl = Kephalophosphoric Acid
Glyceryl )
but no nitrogenised nucleus, and by further chemolysis may split
up into the acids obtained by its side.
Kephalk Acid and Third Fatty Acid, and their Barium Salts. —
The alcoholic solution, from which kephalophosphate had been
precipitated by lead acetate, was distilled to about 300 cc. This
liquor, on cooling and standing, deposited an oil, which was
isolated, and found to float on water ; but on boiling, it became
viscous and solid in the hot. Oil and liquid were treated with
ammonia to strong alkalinity, whereby a white emulsive solution
was produced. To this, water and watery acetate of lead were
added, until, and as long as, a precipitate was' produced. This
was white, adhesive and bulky. It was filtered, washed, and
PHOSPHOPJSED PRINCIPLES OE PnOSPHATIDES.
75
dried, powdered, dissolved in ether, filtered, decomposed with
hj^drochloric acid and water ; the ether solution was washed,
filtered, and when clear distilled to small bulk. The red solution
was treated with w-atery ammonia, filtered, barium chloride added,
when a bulky precipitate was obtained. This was washed for a
long time, until washings were nearly free from, barita ; the pre-
cipitate was dried in air, and treated with ether ; a coloured salt
dissolved and a white salt remained undissolved.
Coloured salt, soluble in ether = Kephalate of barium.
The kephalate of barium dissolves rapidly and abundantly in
ether, and is precipitated from this solution by absolute alcohol.
It is always of a dark colour, which intensifies in the ether, pro-
bably by oxidation. The salt is insoluble, or but little soluble,
in boiling alcohol, and is not deposited from this solution as oleate
of barium is. The acid cannot be decolorised by animal charcoal,
either when in the state of sodium salt, or when in the free state
dissolved in boiling alcohol, and all operations seem only to assist
in farthering its oxidation. It seems that its radicle, while in
the kephalin, is the principal cause of the assumption of colour
and fluorescence by that principle.
White salt, insoluble in ether.
This barium salt appears very much swelled in ether ; when
dry it is white and pulverulent. Hydrochloric acid and water
extract the barium, and washing with water leaves the acid
soluble in ether ; the ether distilled off leaves the acid as a coloured
soft mass, which fuses at 26^, congeals at 25°, and crystallises in
rosettes like margaric acid. It dissolves easily in absolute alcohol,
forming colourless fluid ; a few drops of water added to alcohol
cause acid to separate as an oil on top of spirit; on standing,
colourless rosettes of crystals form in this oil, and may be
separated. The spirit deposits white clouds of the acid.
This acid differs from oleic by its barium salt being entirely in-
soluble in boiling alcohol. It differs from stearic by its lead salt
being soluble in ether. It is not fluid at ordinary temperatures,
])ut semi-solid, and fuses only at 26°, and from concentrated
spirit it is entirely separated and crystallises, while oleic acid
remains dissolved in such spirit, and does not crystallise at such
temperatures.
In the manipulation of these acids in the presence of absolute
76 CHEMICAL CONSTITUTION OF THE BRAIN
alcohol the formation of ethylic ethers, which easily ensues, has
to be carefully avoided.
Glycerophosphoric Ac/d. — The acid filtrate containing that phos-
phorus which was not combined with the kephalophosphoric acid in
the form of glycerophosphoric acid, some nitrogen in an unknown
form, the sodium chloride, together with some free hydrochloric
acid, Avas evaporated to a low bulk, and its acidity carefully
neutralised by caustic soda. Acetate of lead was now added as
long as a precipitate was produced, and the white deposit was
filtered off. It was boiled, filtered, and washed with hot water,
decomposed with H^S, the filtrate neutralised with calcium
carbonate and lime water, filtered and evaporated. When con-
centrated the solution deposited crystalline calcium ghjcero-
2Jliosphate,
Nearin and Second Oily Base. — The solution from which glycero-
phosphoric acid has thus been removed was freed from lead
by H^S, the acetate was decomposed by repeated addition of
hydrochloric acid and evaporation to dryness, and the crystalline
magma was treated with absolute alcohol to extract the hydro-
chlorate of the expected nitrogenised body. To this alcoholic
solution PtCl^ was added, when a slight yellow precipitate ensued,
which was separated by filtration, washed with alcohol, and dried.
It was neurin-hydrochlorate-platinic chloride. It could only repre-
sent a small portion of the nitrogen contained in the chemolysed
kephalin ; as the volatilised vapours had not been collected, some
volatile alkali may have escaped unnoticed. The alcoholic mother-
liquor of this platinum precipitate was freed from platinum by
H.,S. Evaporated, it left a syrupy residue ; this was again treated
with little alcohol and PtCl^, and a slight deposit removed. The
liquor on addition of much ether deposited an oily hody, soluble in
absolute alcohol, reprecipitated by ether. Distilled with dilute
H.,SO_^ and MnO^, it gave a distillate which smelled of acetic acid,
and after neutralisation by soda, was reduced by AgNO^ and by
HgNO.., consequently contained formic acid. These may be con-
sidered as decomposition products of glycerol, or of a body contain-
ing its radicle.
Summary of llesidts of First Chemolysis of Kephcdin. — All first
products are soluble, the soaps imperfectly in water. Hydrochloric
acid precipitates a mixture of fatty acids.
1. Kephalophosphoric acid, consisting probably of kephalic, a
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 77
second fatty acid, and glycerol and phosphoric acid, yet in com-
bination, therefore imperfectly chemolysed.
2. Kephalic acid.
3. A second fatty acid.
4. Glycerophosphoric acid.
5. Ammonia.
6. Glycerin.
7. A base giving oily PtCl^ salt, and being perhaps glyceramin
of a new type. Its solubility in water, alcohol, as hydrochlorate
and as PtCl^ salt is very great, and its precipitation by ether by
no means complete.
(3. Complete Chemolysis of Kephalin by Caustic Soda. — 40 "7 g. of
kephalin, purified by the water solution, filtration, and HCl
process, as before described, were mixed with 5 g. of crystalline
soda hydrate dissolved in two litres of water. The same pheno-
menon was observed during the boiling as in the first experiment.
The boiling was continued during about eighteen hours, of which
three hours took place in water -bath, and the other fifteen hours
on sand-bath, the time taken . to heat it to boiling on the three
days during which experiment lasted not included. While on
the first and second day the mixture had set into a jelly, it did
after the last boiling not become gelatinous again, but remained
a somewhat turbid thick fluid. To this HCl was added until the
precipitate was curd}? and the fluid strongly acid. The precipi-
tate was separated by the filter. It could be washed but im-
perfectly, as after consolidation on the filter, the precipitate set
into a gelatinous tremulous solid mass which allowed no washing
water to pass. This mass of acids was placed into a wide-
mouthed bottle, shaken with water, and treated with caustic
ammonia. It formed a complete solution, "which was filtered on
hot funnel and left no trace of residue. The solution was opaque,
and on agitation showed the silky clouds common to soaps. To
this solution acetate of lead was added until the precipitate was
curdy, and the liquid distinct and filterable. It was now passed
through French filtering-paper, and the precipitate washed* with
much water, being agitated constantly with a glass rod, so that all
parts were well penetrated by water. When the filtrate was free
from chlorine and from lead (from excess of acetate), the precipi-
tate was allowed to drain on paper and a cloth and dried. It
took many days to dry, being frequently crushed and stirred.
78 CHEMICAL COmTITUTIQJSf OF THE BRAIN.
Ether' extracted a coloured body, and left a white salt insoluble.
This was separated by filtration, and washed by being twice
removed from filter, and shaken in a bottle with ether. Filtration
was always difiicult, owing to the finely divided state of the salt.
The white Ph Salt insoluble in Ether was dried in the vacuum, and
after preliminary analysis was further transformed into barium
salt by HCl, water, and ether, solution of acid in NH^HO, and
precipitation by BaCl^. The salt was extracted by boiling
alcohol, and what remained insoluble was analysed. It was found
to be principally stearate.
The coloured Ph Salt soluble in Ether was red in transmitted light,
and fluoresced green, altogether appeared like a solution . of
kephalin. It was concentrated by distillation, and the solution
precipitated by absolute alcohol ; a viscous salt fell down, which
became hard in alcohol, was drained and dried in vacuo. After
preliminary testing, it was transformed into Ba salt by HCl, water
and ether ; solution of acid in NH^HO and precipitation with
BaCl2 ; solution in ether and precipitation by alcoriol. It was
kephalate.
Treatment of the Filtrate containing Glycerophosphork Acid and
Ammonium Base. — It was neutralised with barita water, of which
a slight excess was added. A saturated solution of lead chloride
in water was now added, which produced a flocculent white pre-
cipitate of glycerophosphate. The advantages of the lead
chloride were that no new acid was introduced into the fluid, from
which the ammonium base had yet to be extracted. The fluid
became slightly acid, and was corrected by barita water cautiously
added. When all was precipitated the glycerophosphate of lead was
filtered off", washed, and dried. The mother-liquor was concen-
trated, and then gave another not inconsiderable precipitate with
lead chloride solution. The reaction was continued until a
filtered sample of liquid remained clear with the chloride, and the
second precipitate was isolated and united with the primary one.
The previously feebly alkaline liquid was now feebly acid ; it was
evaporated to a low bulk and then to dryness. It was now dis-
solved in a minimum of water, and precipitated with absolute
alcohol, until this reagent produced no further turbidity, and no
deposit on standing twelve hours. The alcoholic extracts were
united, concentrated, and precipitated with PtCl^. A yellow pre-
cii)itate fell, which will be described further on.
PHOSFUmiSED PRINCIPLES OR PHOSPHATIDES. 79
^ The:.GlyceropJiosphafe of Z^^c? "dried to a hard slightly coloured
mass. (This hardening of glycerophosphates is also observed upon
barium ^altj and >he condition must be borne in mind when it is
subsequently intended to decompose these salts.) It was re-
peatedly crushed, ultimately powdered, and dried in water-oveh.
The total weighed 7*4435 g. Now assuming the 40-7 g. kephalin
to have contained (at 4*2 per cent. P) 1*7 g. phosphorus, then this
corresponds to 19 '38 g. glycerophosphate of lead, of which theory
is—
Acid. Pb salt. Pb pyrophosphate.
3C 36 36 —
9H 9 7 —
P 31 31 2P 62
6 0 96 96 7 0 112
172 Pb 207 2 Pb414
377 588
But as only 7*4435 g. were obtained there is a deficiency of
11*9365 g. Thus much was therefore decomposed further into
phosphoric acid and glycerol.
In order to ascertain approximately the purity of the lead
glycerophosphate, 0*9110 g. was burned until white, and left
0*7440 g. Fh.,V.p^, while theory required 0*7104. The white
residue was treated with acetic acid and found insoluble in it,
which corresponds with pyrophosphate.
The lead salt was now decomposed with hydrothion, the filtrate
treated with CO2 in the cold to expel H2S, and then with milk of
lime to alkalinity, filtered and treated with some CO^. The
filtrate after twelve hours' standing was evaporated near the
boiling-point, and filtered hot from the white precipitate of calcium
glycerophosphate. This when dry weighed only 0*727 g. Of this
0*3400, after strong ignition with HNO3, left 0*207 g. residue,
equal to 60*8 per cent. Csi.^P.fl^. Pure glycerophosphate should
leave 60*5 per cent, of pyrophosphate.
Acid Glycerophosphate of Calcium. — The aqueous filtrate from
which the above salt had been removed whilst hot, was mixed
with three volumes of alcohol, when a light bulky precipitate
ensued. This was isolated by filtration, washed with alcohol and
dried. It weighed 0 526 g. Of this 0 2446 g. gave 0*1250 g. pyro-
phosphate, equal to 51*1 per cent, residue. This corresponds to a
80
GHEMICAL CONSTITUTION OF THE BBAIN
salt which might be obtained from acid glycerophosphate of calcium
by combustion.
CgH.CaPO, + CaH^POg = CgHieCaPPi^ = acid glycerophos-
phate of calcium.
This on combustion may lose C^Hj^Og, and leave H.j^diVjd^, or
half-saturated acid pyrophosphate of calcium, or a body isomeric
with it. By this theory 56*54 per cent, residue should be left,
which differs so much from 51 '1 per cent, found, that we must
suppose the salt to have been one of those peculiar alcoholo-
hydrates which salts of glycerophosphoric acid are prone to form,
as will be specially proved lower down. There also the actual
production of the acid calcic glycerophosphate, here for the first
time theoretically assumed, will be proved by further experiments
and analyses.
The quantity of glycerophosphoric acid lost in the process of
transformation is enormous, and this is invariably observed in all
experiments which I have made.
The Flatinic Chloride Precipitate. — The yellow precijiitate was
washed with absolute alcohol. On drying it became horny,
crumbled up, was viscous outside and discoloured, and ad-
hered strongly to the paper. Water restored yellow colour
and pulverulence. It dissolved in hot water, of which much
was required. On cooling, brilliant small crystals were de-
posited amounting to 0*9428 g. This by analysis proved to be
almost pure ammonium salt, containing only a trace of potassium,
which raised the residue left on combustion to 45*56 per cent.,
while 44*36 per cent, are required by pure NH^ salt. By evapor-
ation over H^SO^ two further small crops of ammonium salt were
obtained ; the rest of the solution dried up to a thick liquid and
did not crystallise.
The filtrate from the PtClj^ scdt was mixed with a large excess of
ether until this produced no further turbidity. An oihj matter
settled, which was purified by repeated solution in alcohol and
precipitation by ether. It was not analysed, as there were no
guarantees of its purity. It was distilled with H^SO^ and
MnO.„ and yielded an acid distillate, in which, after neutralisation
with soda, the presence of formic acid was signalised by the usual
tests.
This platinum salt, which from the reactions detailed may split
up into glycerol and ammonia, reduces the platinum rapidly when
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 81
left in contact with ether- alcohol, and becomes black. Altogether
this product is one of the most difficult matters to treat, and par-
ticularly as it is only obtained in small quantities.
7. First Chemolijsis of Kejjhaliti with Barita Hydrate. — -The
kephalin was prepared by the HCl process, and well washed with
water. The quantity used was 27 "8 gr. It was placed in a flask
connected with a condenser, and a solution containing 80 gr.
barium hydrate was added. The mixture was boiled during
five hours on a sand-bath, when the precipitate became adhesive
and flask cracked. After cooling and filtering the residue was
twice boiled with water, during which it became quite soft and
semifluid ; it was again thrown on a filter and washed to neutrality.
The washings were united with the first filtrate.
The barium salts being drained of water, were rubbed in a mortar
with ether, and then shaken in a bottle with much ether. The
ether dissolved a coloured salt, leaving a tchite salt undissolved, from
which the solution was separated by filtration.
The insoluble ivhite salt was dried in air ; later in -s'acuo. It was
stearate.
The soluble coloured salt had the appearance of kephalate, was
red, and fluoresced green. The solution was concentrated and
precipitated by absolute alcohol. The precipitate was dried in
vacuo, and on subsequent analysis was found to be kephalate.
The liquid containing the glycerophosphate and ammonium base was
treated with carbonic acid until no more precipitate was produced,
filtered, and the filtrate concentrated on a water-bath nearly to
dryness. During this evaporation it did not deposit any salt, as
does the lime salt. The viscous mass was diluted with a little
water to fluidity, placed in a bottle, and mixed with absolute
alcohol until no further precipitate was produced. The precipi-
tate was filtered off" and washed with alcohol.
The precipitate consisted of barium glycerophosphate. It was
bulky, white, and granular, probably alcoholate-hydrate. On
standing, it contracted, and became horny, transparent, and partly
fused. Placed in a glass dish, it fused entirely in a few days,
and then dried to a brittle mass.
6
82 CHEMICAL CONSTITUTION OF THE BRAIN
Sammamj of Analyses :
C
H
Ba
P
0
12-611
2-933
40-950
9-266
34-240
100-000
Formula. — C3H.BaP0^,H,0.
The alcoholic solution from which the glycerophosphate had
been removed was tested,but the products, being small in quantity,
"vyere not analysed. Analyses of this part of the products of the
chemolysis of kej^halin are given in the following experiment.
d. Second Chemolysis of Kejjhalin by Barita. — In this experiment, for
which a compound of kei^halin with cadmic chloride was taken,
two sets of barium salts were again obtained, one soluble, the other
insoluble in ether. The salt which was soluble in ether furnished
a lead salt, which was likewise soluble in ether (28*056 per cent.
Pb), and insoluble in, and precipitated by, ether-alcohol. The
salt which was insoluble in ether, when transformed into lead
salt, was found to consist of two compounds, one insoluble in
ether and in alcohol, the other soluble in ethei^ but precipitated
from it b}^ the addition of alcohol.
The baritic solution was freed from excess of barita hy car-
bonic acid, and the filtrate, after evaporation to a low bulk, was
treated with absolute alcohol. A precipitate of barium glycero-
phosj^hate was obtained, and identified by quantitative analysis.
The alcoholic mother-liquor was neutralised with hydrochloric
acid, and precipitated with jjlatinic chloride. The platinum salt
so obtained was recrystallised from water, and ultimately obtained
in brilliant plates and needles.
Comjmtation of Analyses :
Percentage. -^At Wgts.
-^Pt-=1.
' C 19-8U1 1-650
H 4-726 4-726
N 4-404 -314
O 4-701 -293
Pt 32-219 -163
CI 34-149 -961
10-1
29-0
1-9
1-8
10
59
= (C,Hi,NO),(HCl),PtCl,.
Neurin hydrochlorate i)latinic chloride.
FHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 83
Besides this salt, there was obtained from the alcoholic mother-
liquor from which it had been originally precipitated, a smaller
quantity of a second crystallisable platinic salt. After recrystal-
lisation from water it gave the following numbers on analysis : —
Computation of Analyses :
Percentages. -f-At Wgts. H-Pt = l.
C 9-158 -763 4-1
H 3-134 3-134 16-8
N 5-418 -387 2-0
0. 6-275 -392 2-1
Pt 36-718 -186 1-0
CI 39-297 1-107 5-9
- (C2H.NO),(HCl)2PtCl4.
This salt might be considered as dimethylamin, in which the
third atom of hydrogen is replaced by hydroxyl, or as oxethylamin
simply.
CoH-O
H VN
H j
that is to say, a body formed from neurin, by the loss of three
radicles of methyl and one of water.
A third base was also obtained ; it was soluble in alcohol as
platinic salt, giving the solution a brownish-red colour, and on
the addition of much ether w^s precipitated. It was purified by
repeated solution in alcohol and reprecipitation by ether. Several
grammes of this body which was precipitated in an oily state, but
became solid on drying, were obtained. It was dried at 100° C.
and analj^sed.
Comjndatlon of Analij es :
Percentages.
--At Wgts.
-Pt.
c
12-019
1-001
5-0
H
3-070
3 070
15-3
N
5-019
-358
1-79
0
4-255
-265
1-3
Pt
39-466
-20
1-0
CI
36-171
1-018
5-0
leading to a formula C5H^4N20,HCl,PtCl4. This body, so ano-
malous in its constitution, has probably been derived from neurin
molecles by duplication and subsequent decomposition. Such a
condensation is observed upon the neurin molecles under the
influence of oxidising agents.
C— 2
84 CHEMICAL CONSTITUTION OF THE BllAIN.
i. Third Chemolysis of Kephalin by Barita. — The process was the
same as in the former experiment. The platinum salt gave on
analysis the following results :
Comjmtatlon of Analyses :
Percentages.
H-At Wgts.
-^Pt = l.
c
19-981
1-665
10-3
H
4-719
4-719
29-3
N
4-735
•338
2 0
0
4-443
•278
1-7
Pt
31-832
•161
1-0
CI
34-290
•966
6-0
= (C,Hi3N0),(HCl),Pt01,.
^. Fourth Chemolysis of Keyhalm by Barita. — This experiment was
undertaken upon a larger amount of kephalin than the previous
ones. The kephalin was first warmed in water, whereby it swelled
and became a paste, which was then boiled with two molecles of
BaH^Oo in the ordinary way.
The same barium salts as those obtained in the earlier experi-
ments were again found, and it was moreover observed that one
of the fatty acids contained in them gave the purple reaction,
with sugar and sulphuric acid, which resembles Pettenkofer's test
for biliary compounds.
From the solution drawn off from the barium salts of the fatty
acids the excess of barium was removed by CO.,, and the resulting
filtrate concentrated by evaporation, and then treated with alcohol.
The glycerophosphate of barium thus obtained was converted into
lead salt, and the lead salt into calcium salt (C3HX^aP0^3).
Tlic alcoholic niother-li/juor was freed from alcohol by evaporation,
and the resulting solution after addition of nitric acid was preci-
pitated by i)hospho-molybdic acid, and the product decomposed
by hot BaH.,02. The yellow filtrate freed from the barium which
admitted of removal by CO.j, did yet contain barium in combina-
tion. This was proved by coml)ustion of the dried salt and
analysis of the residue.
The concentrated solution of the base was freed from Ba by an
equivalent amount of very dilute sulphuric acid, then neutralised
by hydrochloric acid, and preci})itated after concentration by
alcoholic PtCl^. The i)recipitate Avas recrystallised from water,
10 gr. being obtained in the shaiie of crystallised i)latcs. The
salt was dried and analysed.
PHOSPHOPJSED PRINCIPLES OR PHOSPHATIDES. 85
Computation of Analyses :
Percentages.
-f-At Wgts.
-=-Pt = l.
c
19-47
1-622
9-7
H
4-94
4-94
29-7
N
4-04
•288
1-7
0
4-36
•272
1-6
Pt
32-85
.166
1-0
CI
34-34
•967
5-8
= {C,Hi3N0)„(HCl),PtCl,.
The alcoholic mother-liquor from which this salt had been pre-
cipitated gave by precipitation with ether a quantity of the fluid
oily salt, the analysis of which has been given in a former para
graph, (C;^Hi^NoO)HClPtCl^, but it was very small in amount.
A third base was also obtained in small quantity by simple
addition of much nitric acid to the original solution, after removal
of glycerophosphate, and before precipitation with phospho-
molybdic acid, and which gave the various alkaloidal tests.
Fatty Acids produced in the Chemolysis of Kephalin from Barium
Salt Insoluble in Ether. — The barium salt insoluble in ether was de-
composed, after complete extraction with ether and alcohol, by
boiling with hydrochloric acid. The liberated acid which solidified
upon cooling was dissolved in ether, washed with water so long
as it removed colouring-matter, and the ethereal solution was
distilled to dryness. The acid which now remained was dissolved
in warm ammonia- water, filtered hot and precipitated with acetate
of lead. The lead salt after being dried was extracted with much
ether, which efi'ected a separation into two salts :
A white lead salt insoluble in ether.
A coloured lead salt soluble in ether.
The Lead Salt Insoluble in Ether. First Preparation. — It was
decomposed with tartaric acid in presence of ether and water,
and the ethereal solution of acid after washing was treated with
animal charcoal, which clarified and partially decolorised it.
The acid obtained by distillation of the ether was repeatedly
crystallised from watery alcohol. Finally four crystallisations
were obtained, dried by fusion for some time at 105^ C, and
their melting-points determined. No. 1 fused at 68' ; 2, at 66' ;
3, at 62-5'; 4, at 63-5° C. These preparations were united
and recrystalhsed from dilute alcohol with animal charcoal.
They finally gave one large crop of acid melting at 68' C, from
86 CHEMICAL CONSTITUTION OF THE BRAIN.
the mother-liquor of which a small further quantit}^ was obtained,
melting at 66° C. It w^as stearic acid, mixed with a small quantity
of a lower homologue.
Second Preparation. — Another portion of lead salt was decom-
l^osed with tartaric acid and ether, and the ether distilled to a low
bulk. On cooling, a large crop of nearly white crystals Avas
obtained. After separation from the highly coloured mother-
liquor l)y filtration and pressing, the acid was twice recrystallised
from dilute sjnrit with animal charcoal. There was thus obtained
a perfectly colourless acid, having a fusion-point of 70' C. This
was dried for analysis by fusion at 110° C, after which treatment
its fusion-point was found still the same. Analysis led to the
empirical formula CjgHgyO^,, which is that of stearic acid. This
acid has the same melting-point, microscopic appearance, and
other physical characteristics as stearic acid. A portion of it
dissolved in dilute ammonia gave a salt w^hich dissolved perfectly
on heating, almost completely separated in crystals on cooling, and
otherwise exactly resembled stearate of ammonium prepared from
pure stearic acid.
Third Preparation. — Obtained by chemolysis with hydrochloric
acid. The insoluble in ether lead salt, from insoluble in ether
barium salt obtained in this chemolysis (' Keport,' 1876, p. 118),
was examined as to its identity with the analogous salt obtained
in the barita chemolysis. It was decomposed with tartaric acid
in presence of ether, and the ethereal solution was distilled, and
when sufficiently concentrated was allowed to crystallise. The
crystals, purified by recrystallization from watery alcohol with the
aid of animal charcoal, gave a white product, which fused at
69° C. and was analysed. In all respects it was identical with
stearic acid.
Lead Salt Soluble in Ether. — The clear ether solution was distilled
to dryness, again dissolved in ether, decanted from matter rendered
insoluble, and treated with a concentrated solution of tartaric acid.
The ethereal solution of the acid free from lead gave on evapora-
tion a viscid mass which, being dissolved in ammonia and pre-
cipitated with barium chloride, gave a viscous barium salt. This
yielded to boiling alcohol a small amount of a salt which was
analysed. It contained 19-65 per cent, of barium. This body
requires much further study.
Barium Salt Soluble in Ether, Keplialate. Product of the first
PHOSPHORISED PIUNGIPLES OR PHOSPHATIDES. 87
Clismolysis. — This salt dissolved in ether with a red-brown colour,
from which it was impossible to free it by any process whatever.
To try whether this colour were due to oxidation caused by contact
with the air, some of the free acid was enclosed in a tube with a
measured volume of oxigen gas, but no absorption was observed.
It is therefore clear that any oxidation must take place in
ethereal solution, and in that case must be ascribed to the per-
oxide of hydrogen formed by the ether. The red-brown colour
appears to be proper to the acid and its salts. To ascertain
whether the soluble in ether barium salt contained more than
one acid, it was decomposed with tartaric acid in presence of
ether, and the liberated acid obtained upon distillation to dryness
was dissolved in ammonia and precipitated with acetate of lead.
The lead salt was dissolved in ether, and fractionally precipitated
in three portions by successive additions of alcohol. The fractions
were analysed, and gave 38-07, 38*48, and 36-39 per cent. lead.
It is therefore probable that the soluble barita salt mainly
consists of one acid only. The following attempt was made to
obtain it in a state fit for analysis.
After the barium salt had been exhausted with boiling alcohol
it was dissolved in ether, and the intensely coloured solution pre-
cipitated by alcohol ; the precipitate was redissolved in ether and
again precipitated. The mother-liquors removed no colouring-
matter. The compound was next decomposed by tartaric acid,
the free acid dissolved in ether ; the solution was distilled to dry-
ness, and the residue extracted with absolute alcohol. The
solution thus obtained was treated w^ith . ammonia in excess, and
the filtered clear soap solution precipitated with acetate of barium.
The precipitated salt was analysed.
Percentages.
-^At. Wgts.
--Ba-=1.
0
59-556
4-963
37-3
H
8-488
8-488
63-8
Ba
18-28
•133
1-0
0
13-673 >
•854
6-4
These data lead to a formula of about Ba(Cj^^H3^03)2.
/. Product of the Secondary Cliemdysis with Barita a7id Caustic Soda
in Succession. — On account of these unsatisfactory results, and from
a fear that the salt might contain traces of undecomposed kephalin,
it was again submitted to a barita chemolysis ; but as it agglo-
merated into large masses, into the interior of which the barita had
88
CHEMICAL CONSTITUTION OF THE BBAIN
little access, it was decomposed by treatment with hydrochloric acid
and water. The free acid after solution in ether and distillation
to dryness was dissolved in warm dilute caustic soda, a large
excess of soda added, and the whole boiled seven or eight hours
a day for several successive days. There was great frothing
which could not be prevented, but any loss was obviated by
allowing the froth to issue from the wide beak of the platinum
retort employed, into a large beaker where it slowly subsided.
From time to time the fluid which collected was returned to the
retort. At the end there was obtained a brown turbid solution
showing on agitation the silky clouds common in soap solutions.
It was filtered as clear as possible by the vacuum method, and
the solution precipitated with acetate of lead. The voluminous
precipitate, after washing and drying, was extracted with ether,
when it mostly dissolved, leaving, however, an amount of insoluble
salt, probably stearate. The clear ether solution was treated with
a concentrated solution of tartaric acid, the ethereal solution
of acid separated from tartrate of lead, distilled to dryness, dis-
solved in a minimum of caustic soda, and precipitated with barium
chloride. The barium precipitate was washed, dried, suspended
in ether, and a solution was separated by decantation and filtra-
tion from a white insoluble salt which appeared much swollen in
ether. The insoluble salt was analysed, but did not lead to any
formula. The ethereal solution after concentration was preci-
pitated by addition of a minimum of absolute alcohol and the
precipitate analysed.
Percentages.
-^At. Wg-ts.
H-Ba 1
c
58-55
4-878
34-61
H
8-45
8-45
60-01
Ba
19-29
•1408
1-00
0
13-71
•8569
6-08
leading to the formula Ba(Cj^H3Q03)^.
The filtrate on examination was found to contain a small
quantity of a kephalin-like body. The precipitate was therefore
again dissolved and reprecipitated by a little alcohol, washed with
absolute alcohol, and analysed :
Percentages.
-^At. Wgts.
-f-Ba = l
c
57-82
4-818
33-20
H
8-14
8-14
5610
Ba
19-89
•1451
1-00
0
14-15
•8842
6^09
'pointing to an
empirical formula Ba(Cj;Hos.O;.)j.
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 89
The acid obtained by decomposing this salt with hydrochloric
acid is a dark-coloured viscid oil at the ordinary temperature,
which is wholly soluble in alcohol, the solution not being de-
colorised by even large quantities of animal charcoal. On
evaporation, or on addition of water, the acid separates from the
alcohol in brown oily drops. On fusion with potash, no solid acid
is obtained, but a brown acid which has all the properties of the
original body, and gives a barium salt which contains about 19
per cent, of barium, and is soluble in ether.
Thus this acid gives none of the ordinary physical guarantees of
purity, but the pertinacity with which it retains its composition
and properties under the most varied and severe treatment, points
distinctly to its chemical unity, while the quantity in which it
occurs shows its radicle to be a principal ingredient in the
kephalin molecle.
yt. Theory of the Chemical Constitution of the Kephalins. — Accord-
ing to the theories hitherto in vogue, kephalin may be regarded,
considering its elementary composition and the products of its
chemolysis, as a body in which two hydroxyls of the glycerin
molecle are replaced by fatty acids, and in which the third
hydroxyl is replaced by phosphoryl, which latter in its turn has
one hydroxyl replaced by an ammonium base, thus :
( Fatty acid A.
C3H5 \ Fatty acid B or C.
( H0(0P0)C5Hi,N0.
The whole of the phosphorus is apparently contained in the
radicle of glycerophosphoric acid, and in no other form. This
body is first obtained on chemolysis, but being somewhat un-
stable, it is not difficult in the presence of bases to decompose it
further into glycerin and phosphate of the base used.
The whole of the nitrogen appears to be present as neurin ; the
other bases which have been obtained being probably derived
from neurin by secondary changes. This secondary decomposition
would explain why the amount of the neurin obtained from a
given weight of kephalin is less, sometimes much less, than the
amount which theory would lead us to expect.
Of the fatty acids contained in kephalin, various barium, lead,
and magnesium salts have been examined and analysed. It is
highly probable that the acid contained in the main soluble in
ether, barium, or lead salt, namely kejjhalic acid, belongs to a
90 CHEMICAL CONSTITUTION OF THE BRAIN
series of acids containing at least one atom of oxigen more than
the ordinary fatty acids. It is this fatty acid which impresses
its pecuHar character upon all the kephalins, and without its
presence a phosphorised body constituted as above assumed does
not seem to exhibit the properties of a kephalin. But the
kephalins may vary as regards the second acid ; in the principal
kephalin this acid is stearic; but in certain kephalins which occur
in subordinate quantity the second acid is either an acid of lower
fusing-point than, though probably homologous with, stearic acid,
or an acid not homologous with stearic, and giving a lead com-
pound soluble in ether. The constitutional formula of the
jH'incipal kephalin, or lephalo-stearo-neuro-glyceropliosphate would
thus be the following :
A kephalin with palmityl, C^^Hg^O.,, in place of stearyl would have
the summary formula C4jH^-NP0i^ ; a kephalin with margaryl,
Cj^HygO^, would be C^^H^c^NPOg. If there were several homolo-
gous kephalic acids such as some analyses seem to indicate, then
for a kephalyl of formula Cj^Hy^^Oy combined with either stearyl,
or margaryl, or palmityl, the foregoing formulae w^ould have to be
increased by CH^ each, so that the most complicated kephalin
might contain 44 atoms of carbon.
It will be seen that none of these hypotheses explain either the
deficiency of hydrogen or the excess of oxigen in the various
kephalins and their compounds which have been analysed. This
discrepancy can only be eliminated by further researches carried
on by the light of those given in the foregoing.
We have considered the kephalins as glycerides, in which three
hydroxyls are substituted. It is, however, evident from the con-
stitution of sjiliingomyelin, to be described l)elow, and generalised
in the introduction, that they may also be considered as plm-
jyJiatides, or bodies held together by phosphoric acid, thus :
Phusphoik Acid. Keplialhi.
the neuryl re})lacing an hydroxyl in glyceryl, and being the
9
I HO
OP ' HO OP
I HO
PHOSPHORISED PPJNCIPLES OR PHOSPHATIDES. 91
radicle which is the earliest to be detached by chemolysis. On
this assumption the kephalophosijhoric acid above described would
have the formulae :
OP C,3H3A -CasH.oPO,.
3. Paramyelin : ITS Isolation, Analysis, and Compounds.
Pararmjeliti is a nitrogenised phosphatide, and is in the first
stage diagnosed and separated by the solubility of its cadmium
chloride compound in hot benzol, from which solution, filtered
boiling, it is deposited on cooling. In this condition the com-
pound is very voluminous and gelatinous, and is only with ver}^
great difficulty separated from the lecithin cadmium chloride
compound which remains in solution in the cold benzol. The
mechanical aids to this separation are two in number ; one the
cylindrical vacuum filter described, the other the use of very
large volumes of benzol for the extraction of the soluble com-
pound, the removal of the solution by the syphon from above
the deposit, and very frequent repetition of this process. In this
i:)rocess the large volumes of benzol require many distillations for
recovery ; the deposition of the paramyelin compound requires
time. But when once obtained free from matters soluble in cold
benzol, the compound behaves with great precision. It is, whilst
moist with benzol, washed with spirit, which dissolves any colour-
ing-matter, and it is well to repeat this washing by shaking the
comjDound in a bottle, etc., until the supernatant spirit is colour-
less. The compound thus obtained is soluble in boiling spirit,
and deposited on cooling ; it is easily decomposed by hydrothion
in sj)irit, and the hot solution yields on cooling white crystallised
paramyelin hydrochlorate. This by re -crystallisation or washing
loses its acid easily, and free crystallised paramyelin remains.
A specimen of paramyelin caclm'iuin chloride compound obtained
from the extracts of human brain by the process described under
lecithin, but without the previous purification by lead, soluble in
boiling, insoluble in cold benzol, dried at 100°, gave on analysis
13-70 per cent. Cd, 3-59 per cent. P, 9-68 per cent. CI (this
requires 15-27 per cent. Cd, whereas only 13*70 per cent. Cd were
found, total CdClg calculated from CI = 24-95 per cent.), 1-69 per
cent. N, 47-19 per cent. C, and 7-94 per cent. H.
92 CHEMICAL CONSTITUTION OF THE BRAIN.
These data exhibit the following ratios :
P : N = 1 : 1-06
P : CdCl, = 1 : 1-20
N : CdCi; = 1 : M3
If CdCl^ be calculated from CI = 24-95 \
then the organic molecle is = 75-05 j
If CdCl^ be calculated from Cd = 22-39 )
then the organic molecle is = 77-61 }
= 100
= 100
These relations thus show a slight excess of nitrogen over the
relation N : P = 1 : 1, and a more important excess of cadmium
chloride over the presumable relation N : CdCl., = 1:1. Bat
considering that the compound is a first product, the relations
are satisfactory, as showing the firmness of the compound. In
the following I give a comparison of this salt with the paramyelin
salt from ox first described.
Summary of Hitman Paramyelin Summary of Ox Paramyelin
Cadmium Chloride. , Cadmium Chloride.
Percents. Percents.
C 47-19 N C 49-288
H 8-299
75-05 N 1-598 V 78-725
H 7-94)
N 1-69 >-7
P 3-59 \
O 14-64
(Calcd.) Cd 15-27 1 (Calcd.) Cd 13-020 )
(Found) CI 9-68 / (Found) CI -255 ) ^'"^
P 3-396
0 16-144
Computation of the Organic Moleclcs of these Salts.
Percents ^ by At. Ws. -r- P = l. Percents. -^ At, Ws. -J- P = l.
408 C 62-87 5-239 34 C 62-607 5-217 37-5 456
68 H 10-58 10-58 68 H 10-541 10-541 75-8 76
14 N 2-25 0-160 1-03 N 2-029 0-144 1-0 14
31 P 4-78 0-154 1 P 4-313 0-139 1-0 31
l-_8 0 19-50 1-24 8 O 20 510 1-281 9*2 144
649 ' 721
The percentages of carbon and hydrogen are practically iden-
tical in both organic molecles. Other data show distinctly that
the salt is not, and does not contain, either amidomyelin or
sphingomyelin. The absence of lecithin follows from the solu-
bility in cold benzol of its cadmium chloride compound. The
data, however, do not show that the compound is unitary, and
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 93
does not contain two or more similar principles (paramyelins).
They also do not sho w the exact composition of the organic
molecle, for, as we know from a vast amount of experience, this
can only be ascertained with the aid of several compounds, and
of the decomposition products of the principle under the influence
of chemolytic agents. For the study of these relations the past
has afforded no time or opportunity, and it must therefore be left
to the future.
Preparation of Free Parainyelin from the CaJmiam Chloride
Compound.
The CdCU salt of human paramyelin was suspended in spirit,
and treated with H2S, at first at the ordinary temperature, later
on at 75° in the water-bath, until it was completely decomposed.
The filtrate, on standing and cooling, deposited white crystallised
paramyelin. The crystals were rhombic and hexagonal plates of
microscopic dimensions. They were collected on a filter, washed,
pressed, recrystallised from spirit to remove a trace of colour and
the rest of the hydrochloric acid, and dried in vacuo. On analysis
they gave 4-31 per cent. P, and 2*06 per cent. N.
These two analyses show that N : P = l-00 : 1-00.
The atomic weight of j^aramyelin as deduced from the per-
centage of phosphorus is 688 ; as deduced from nitrogen, 666 ;
mean, 677 (for the free body). The atomic weight of paramyelin
(human) as deduced from the organic molecle in the CdCl., salt is
649 ; that for ox paramyelin combined in the same manner is 721 ;
mean 680. These two means are practically identical.
The question now arises whether this phosphatide contains
glycerol or not, neurin or not ; these questions can be answered
by chemolysis only.
Paramyelin Cadmium Chloride (Ox). — C3gP^-IS['P09,CdC1.2.
From Ox-huttery after Kephaloidin.
"When the buttery matter dissolved in ether had been precipi-
tated by alcohol and the kephaloidin been removed, the mother-
liquor on standing deposited some secondary kephaloidin and
cholesterin. These were filtered off* ; the liquid was precipitated
with CdCl2 ; the precipitate was washed with alcohol, and pressed ;
it was next extracted with ether (which dissolved a small quantity
of kephaloidin CdCU) until pure, dried, and analysed. The result
of the analyses showed that the body was a CdCl., compound.
94 CHEMICAL CONSTITUTION OF THE BRAIN.
The organic molecle amounted to 77 "49 per cent,; the CdCl^ to
22-51 per cent.
Treatment nitli Benzol. — It was found that the compound was
entirely soluble in boiling benzol, and deposited a portion on
cooling which was white and voluminous. Another portion re-
mained dissolved in the cold benzol. The deposit was isolated by
filtration, dissolved once more in boiling benzol, and was deposited
as a swelled gelatinous mass ; from this benzol was drained by
blotting-paper, and the residue was dried.
Faramyelin Cadmium Chloride^ Q.^fi^^VOc^.C^GV^. — Insoluble in
cold benzol. The analyses were carried out in the usual manner,
but the Cd was not estimated.
Sv.mmary :
Hypothetical
Percents.
c
49-288
H
8-299
N
1-598
P
3-396
fO
(16-144
)Cd (13-020)
CI
8-255
100-000
'8-725
21 275
100-000
Computation of Organic Molecle :
Percents. -4-by At. Wgts. -^byP = l.
C 62-607 5-217 37-5
H 10-541 10-541 75-8
N 2-029 0-144 " I'O
P 4-313 0-139 1-0
O 20-510 1-281 9-2
100000
The formula CygHn^NPOg gives an atomic weight of 720, but
the atomic weight calculated from the CdCl^ is only 677 ; 720 + 183
(CdCl2) = 903 requires 20-2 per cent. CdCl^. There is, therefore,
still an irrationality between the chloride and the organic molecle,
in the sense of the metallic salt being in excess.
4. Myelin : its Isolation, Analysis and Compounds.
General Definition of Myelin. — The leading features of the prin-
ciple here to be described will distinguish it with great precision
from all similar matters. When freshly obtained it is white like
LONDON
aCHOOL OF MEDICINE
V*^^. ffOaV^OMEH
PHOSPHORISED PBINCIFLES OR PHOSPHATIDES. 95
bleached ivory, but when kept for some time it becomes a little
yellowish and waxy. It crystallises, from ether or absolute
alcohol solution on slow evaporation, in curved needles aud scales
of a rhombic ovoid shape, which are well seen under the micro-
scope with a power of x 400. When it is in minute crystals it
remains powdery even after drying, but when drying in body
after deposition from alcohol and washing by ether, it becomes
transparent and waxy, cuts like dry walnut kernel, and when dry
can be powdered. The powder is perfectly white. It swells and
emulges with water, particularly on the application of heat, in the
manner defined for all phosphorised cerebral principles. The
solution iridesces bluish-white from polarisation of the minute
particles. This solution gives the reactions to be described. It
dissolves in hot alcohol abundantly, and is deposited on cooling in
white tufts, granules, and masses of peculiar appearance, and on
slow evaporation in crystalline needles. The alcohol retains little
myelin in solution when cold, and gives no precipitates with
CdClg and PtCl^. It dissolves very sparingly in hot ether, and
is almost immediately deposited from this solution when its tem-
perature sinks. It is less soluble in cold ether than in boiling.
It contains more than three per cent, of phosphorus. With Pb
acetate and ammonia it gives a white salt, which is insoluble in
alcohol and ether, and contains an atom of lead. Myelin is conse-
quently a dibasic acid.
Modes of oUaining Myelin. — It can be obtained directly, without
the intervention of precipitants, from the cold alcohol extracts of
white matter, by concentration and cooling, redissolving the pre-
cipitate, and letting the solution stand for a long time in the cold,
when myelin is deposited crystalline. After isolation it has to be
washed with a little ether, combined with lead, and freed from
sphingomyelin by boiling spirit.
The ether extracts of white matter may be precipitated by
alcohol, the precipitate emulged with water, treated with lead
acetate and ammonia, washed, and extracted with hot alcohol and
ether in succession, when ultimately white myelin lead, of the for-
mula to be given, remains insoluble in these agents. The lead
salt decomposed by in water, and the precipitate extracted
with hot alcohol, yields white myelin ; or the lead compound may
be decomposed in hot spirit by hydrogen sulphide.
Differences and Sejmration from other Cerebral Prhiciples. — Myelin
96 CHEMICAL CONSTITUTION OF THE BBAIN
can be separated from kephalin and kephaloidin and allied bodies
by the operator using the peculiarity of its being veiij little soluble
in cold ether, in which these bodies and their compounds are easily
solul)le. The solutions should always be exposed to a very power-
ful freezing mixture, and filtered through a filter and funnel sur-
rounded with freezing mixture. From lecithin myelin can be
separated by the lead process.
Myelin can be separated from the cerehrosides by much boiling
alcohol, in which both are largely soluble, but the cerehrosides are
almost insoluble in cold alcohol, in which, therefore, myelin would
remain dissolved on cooling more readily. But the lead process
is preferable, and necessary in any case as a means of precipitation.
An absolute separation of myelin from the other phosphorised
principles is best effected by the processes employing lead acetate,
and from cerehrosides (as obtained by alcohol process) by ex-
traction with boiling alcohol and separation after cooling.
No phosphatide, however similar in bearing to myelin, should
be assumed to be myelin before it has been in combination with
lead, and been found insoluble as lead salt in boiling alcohol and
in ether.
M//elin Lead.—C^QR^.^'Ph^VO^^y The ether-solution from
white matter, after exhaustion by freezing, was precipitated by
alcohol ; the bulky precipitate was filtered, washed, and dried
in vacuo, and during this process repeatedly pounded in a mortar.
It was now swelled in water, and subjected to dialysis ; it
formed a thick, slimy, gummy or starch-like emulsion, in which
many small crystals of cholesterin formed. The addition of
w^atery Pb acetate produced a dense curd, which separated easily
from fluid ; it was placed on a cloth filter and allowed to drain
away its mother-liquor. The precipitate was placed in alcohol
and warmed, whereby little else but water was extracted (one
litre alcohol left on evaporation to dryness a little brown matter).
More warm strong alcohol noAv extracted much cholesterin. Hot
boiling absolute alcohol extracted 7nuch cholesterin and a little
yellow smeary lead salt. The insoluble part was soft, waxy, but
on cooling granular. It was now placed in ether, whereby a
yellowish fluorescent lead salt of kephalin was extracted. This
latter salt was i)recii)itated by absolute alcohol, deposited as a
yellowish oily body, wdiich l)ecame hard on standing. This has
been treated under kephalin. A ichite pulverulent salt remained
PEOSFHORISED FFJXCIPLES OR PHDSPHATIDES, 97
insoluble in the ether, was thoroughly washed with ether on the
filter, also shaken with ether in a bottle, and again washed on
the filter. It shrunk much on drying. It was insoluble in benzol.
Substance dried at 100^ C. gave, on analysis, data which are
arranged in the following summary of analyses and theories :
Percentages.
-^-byAt. Wgts.
-j-byPb = l.
-f-byX = l.
-^bYP =
C 50-88
4-240
44-63
41-56
40-38
H 7-89
7-890
83-05
77-35
75-13
Pb 19-76
0-095
1-00
0-93
0-90
N 1-44
0102
1-07
1-00
0-97
P 3-28
0-105
1-10
1-02
1-
0 16-75
1-046
11-01
10-25
9-96
100-00
The organic body in the salt = 100-19-76 = 80-24.
Percentages of elements found in organic body and theories :
-^by At. Wgts.
by X = l.
H-by P-1.
c
63-409
5-2840
41-24
4009
H
9-833
9-8330
76-76
74-60
X
1-794
0-1281
1-
0-90
P
4-087
0-1318
1-02
1-
0
20-874
1-3046
10-18
9-89
99-997
There are thus arguments at hand for atomic weights with
from 40 atoms to 44 atoms of carbon ; but the combined metals
and salts are perhaps less to be relied upon for atomic weight
determinations of the phosphorised principles than the consti-
tutional elements P and X. These latter, therefore, prevail in
my opinion as determinants, particularly as they agree well with
each other. I therefore accept C^^jH-.^XPO^q as the formula of
the body combined with lead, and adding 2H in the place of Pb,
the formula of the free body will be C^,3H--XP0j,).
Theory of
Found.
Elements.
At. Wgts.
Percentages.
40 C
480
63-15
63-409
75 H
75
9-86
9-833
1 X
14
1-84
1-794
1 P
31
4-07
4-087
10 0
160
21-05
20-874
760
7
98 CHEMICAL CONSTITUTION OF THE BRAIN.
Decomposition of PI Salt hj H.^S. — A portion was decomposed by
H^S while suspended in ether. The ethereal filtrate from the
PbS deposited a white flaky matter on being shaken, which in-
creased in quantity on standing. It was allowed to go to dryness
spontaneously, and left an abundant white residue, which was
soft, and smelled peculiarly. It fused above 100°, was perfectly
fused about 125° to 130°, and on cooling was quite solid again at
100°. On being heated further it cracked and spirted, then gave
off strong-smelling fumes, burnt with a white luminous flame,
and left a charcoal difficult to incinerate. Fused with nitre and
soda, and the fused mass dissolved in HNO3, the tests for lead
gave negative results, but the tests for P2O3 gave evidence of
abundance ; so that the H^S treatment removed all the lead.
The supposed PbS, when heated, fused, and gave off car-
bonaceous vapours, and behaved in such a manner as to indicate
that it did yet contain much organic matter.
The entire quantity of finely-powdered lead salt was now placed
in absolute alcohol, and decomposed with H^S while being heated
in a water-bath, filtered hot, and extracted with hot alcohol as
often as was necessary to effect complete exhaustion. The alcohol
extract, on cooling, deposited a crystalline mass. This was re-
crj^stallised from absolute alcohol, when a tendency to stearoco-
notise became evident in the deposit ; but all ultimately dissolved
with the aid of hot ether, and the first purest portion of crystals
was analysed. Dried at 100° C, they became coloured on surface.
Summanj and Computation :
Percents. -^byAt. Wgts. -f-byP = l.
C 62-651 5-221 39-0
H 10-340 10-340 77-1
N 2-000 0-142 1-
P 4-170 0-134 1-
O 20-829 1-301 9-7
The isolated myelin thus exhibits the formula C3C)HH.^NP0y.
B. SUBGROCTP OF DINITROGEXISED MONOPHOSPHATIDES.
N:P=-2:1.
1. Amidomyelin: its Isolation, Analysts, and Compounds.
Amidomyelin is met with in certain precipitates obtained from
brain extracts by the agency of cadmium and platinum chloride.
These precipitates are, as regards crystalline appearance and bearing
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 99
towards solvents, seemingly homogeneous ; but on elementary
analj^sis they show an irrationality between the phosphorus and
nitrogen, which in not a few cases assumes the proportion of
P : N = 2 : 3, all other elements being present in nearly the same
average atomic proportions as those in which they are found in
phosphatides in which P : N = 1 : 1. I explain these variations
of the nitrogen as due to the presence of a compound in which
P : N = 1 : 2. After the discovery of ajjomyelin, and lately of
sjjhingomyelin, in both of which principles P : N = 1 : 2, I isolated
amidomyelin by the process.es which have already been partially
described under the chapter relating to lecithin. These processes
had to be guided by incessant quantitative elementary analysis by
which to control the progress and direction of the purification of
the principle sought to be isolated. It was found that differentiat-
ing solvents and combinants were the principal means for effecting
this isolation and purification, and that so-called fractional crystal-
lisation and recrystallisation were only of subordinate value. No
diagnostic value was found to be attached to so-called uniform
crystalline or crystallised appearances ; for a great number of
chemically similar or dissimilar bodies would crystallise in such a
manner as to make the impression of homogeneity upon the eye,
while their diversity could be quickly and incontrovertibly proved
by appropriate chemical reagents.
Process for the Isolation of Amidomyelin.
The buttery matter from human or bovine brains is dissolved
in hot spirit, and to the solution an ammoniacal solution in spirit
of lead acetate is added as long as a precipitate is produced.
This precipitate is removed by filtration on a funnel heated by
steam. It contains kephaloidin and myelin as lead salts ; lead
salts of phosphatides, free from nitrogen, and some lead salts of
cerebrinacides, the latter in small quantity. The filtrate deposits
on cooling a mixture of cholesterin with lead salts, particularly of
myelin and cerebrinacides, and other phosphatides in the free
state, amongst them some amidomj^elin. The clear filtrate is
mixed with a spirituous solution of cadmium chloride as long as
a precipitate is produced : an excess of cadmium solution is then
added, and the mixture is allowed to stand for the precipitate to
contract and settle.
The mixture of cholesterin, lead salts, and other phosphatides,
7—2
100 CHEMICAL CONSTITUTION OF THE BRAIN
is again boiled with spirit and allowed to become cool without
haying been filtered while hot. The liquid is filtered from the
insoluble and crystallised matter, and in its turn treated with
cadmium chloride solution in the manner stated for the first
solution. In this manner the cholesterin and lead salts mixture
is extracted with spirit as long as the mother-liquors give precipi-
tates with cadmium chloride. The last extracts are the richest
in amidomyelin. The cadmium chloride precipitates are all
united, washed with spirit by decantation until the washings are
colourless, exhausted with ether by decantation, dried in vacuo
over oil of vitriol, powdered, and subjected to the benzol process,
whereby they are separated into the three different compounds
which have been described under lecithin. The compound in-
soluble in boiling benzol is the amidomj^elin dicadmium chloride
compound. When the cadmium precipitates are not thrown
together, but treated separately by the benzol process, it is
observed that those obtained from the earliest spirit solutions
contain only little amidomyelin, while this ingredient gradually
increases in quantit}^ until in the cadmium precipitate obtained
from the ninth or tenth solution there is found as much as 40 per
cent, of the salt insoluble in boiling benzol.
Amidormjeliii Dicadmium Chloride Com^joimd, InsohdtJe in Boiling
Benzol from Ox huitery after Lead Process.
Sf/nopsis of Ancdijses :
(1.) (2.) (3.) (4.) (5.) (6.) (7.) (8.)
C 43-78 43-87 — — — — — —
H 7-68 7-72 _ — — —
N — — 2-38 2-55 2-38 — —
P _____ 2-57 2-58 —
Cd — ______ 17-99
CI ________ 11-42
Mean of Ancdyses and Theory of Formvla.
Percents. -^At Wgts. -^P = 1. ^N = l. Theory.
C 43-825 3-652 43-73 42- C,, 528 ]
H 7-70 7-70 92-2 88- H,., 92 |
N 2-43 0-1735 2-07 2- N., 28 |^ 839
P 2-59 0-0835 1- 0-96 P" 31 j
O 14-045 0-8775 10-5 10- O^o 160 J
Cd 17-99 0-1606 1-92 1-84 Cd^ 224 (
CI 11-42 0-321 3-84 3-70 CI, 142 / ^^"^
100-000 1205
FHOSPHORISED PRINCIPLES
C44H3,N,POio(C(iCl2), requires 30-37 per cent. CdCl,.
found 29-41
C44H92N2POioCdCl2 requires 17-90
Preparation of Free Amidom.yelin from the Cadmium Chloride
Compound.
(a) By the Hydrothion Process. — The cadmium chloride salt
described in the foregoing is finely powdered, suspended in spirit,
and the mixture is saturated with hydrothion at the ordinary
temperature. It is then heated in a water-bath until the spirit
boils, while the introduction of the sulphuretted gas is continued
until the cadmium is all transformed into the yellow sulphide.
AVhen a filtered sample of the solution is no longer altered by
hydrothion, the whole is isolated by filtration on a heated funnel.
On cooling it crystallises, the quicker the more concentrated it is.
After twenty-four hours' standing the crystals are collected, washed
with spirit and pressed, redissolved in a minimum of spirit (in
which, before dissolving, they melt into an oil), and again allowed
to crystallise. This process is repeated until crystals and mother-
liquor are both perfectly colourless. In this process there is the
danger that some of the amidomyelin is decomposed under the
influence of the four molecles of hydrochloric acid which are set
free by the decomposition of the cadmium chloride. This chemo-
lysis may yield some fatty acid, which may remain mixed with the
amidomyelin, and is difficult to remove. In consequence, the
carbon and hydrogen of the free body may be found higher than
the theory derived from the salt. The free body also retains some
hydrochloric acid — in the first instance, less than 1 per cent,
(found 0*93 per cent. CI) — which diminishes to a trace by repeated
crystallisation from spirit. But to remove this hydrochloric acid
entirely requires a circumstantial process and the employment of
silver oxide or mercuramin, and, again, hydrothion and frequent
recrystallisation.
(6) By Dialysis. — The cadmium compound is suspended in
w^ater, and placed in a corrugated piece of vegetable parchment,
folded like a plaited filter, and placed inside a funnel. The
funnel is closed with a cork, or some kind of tap, at its lower
aperture, and the space between the funnel and parchment is
filled with distilled water. This is renewed as long as it contains
cadmium chloride, indicated by sulphide of ammonium and nitrate
102 CHEMICAL CONSTITUTION OF THE BRAIN.
of silver. The amldomyelin is at last found to be completely dissolved
in the watei\ and the solution can be filtered clear through the
densest filtering-paper. When this solution is gently warmed, it
sets into a jelly. This latter may be evaporated on the water-
bath, with constant stirring, to near dryness. The residue is
dissolved in hot spirit, the solution treated with hydrothion to
remove a trace of cadmium, and allowed to cool and stand.
White amidomyelin crystallises, to be purified by recrystallisation,
etc., as above described.
{(:) From the Acid Mother-liquors, filtered from the crystallised
amidomyelin, the hydrochloric acid is removed by mercuramin
added in fine powder, with stirring and warming. When the
powder does not any longer change colour, but retains its
canary-yellow tint, all the acid is precipitated. The solution is
filtered warm, and, yet warm, treated with a little hydrothion to
remove the trace of mercuramin which is dissolved in the hot
spirit. It is then concentrated, and allowed to crystallise ; or if
coloured, it is mixed with cadmium chloride solution, and the
washed cadmium chloride compound is treated anew as above
described.
Properties of Amidomyelin.
Amidomyelin crystallises in snowy-white microscopic plates and
needles, arranged in stars and disposed in irregular masses. They
dry in vacuo over oil of vitriol to a perfectly white mass, which is
easily powdered, and can be dried in the water-oven below 100°.
With sugar and oil of vitriol amidomyelin gives the purple of
Kaspail's reaction rather quickly and deeply ; it is at present not
known whether the reaction is due to the presence of the oleyl,
cholyl, or sphingosyl radicle in the molecle of the amidomyelin.
The remarkable solubihty of the freshly dialysed amidomyelin in
cold pure water, and insolubility in slightly warmed water, must
again be pointed out. The change which it undergoes when its
watery solution is warmed is permanent : the jelly j^roduced by
warmth does not redissolve on cooling. This phenomenon is of
great importance in the study of the functions of the immediate
principles in the brain ; it is calling for further investigation, and
comparison with the bearing of the other phosphatides under
similar conditions. One of these, not yet accurately identified,
has the property of being slimy and difi'used and unfilterable in
cold water, while becoming hard and contracted in boiling water.
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 103
so that the water can be filtered off. This substance, with
fresh cold w^ater, gradually resumes the slimy greatly hydrated
condition.
Theory of Amiclomyelin (Ox) as deduced from its Cadmium Chloride
Compound, and Comparison with the Theory of Sphingomyelin
{Apomyelin).
Taking the organic molecle from the analysed CdCU salt, with
29-42 per cent. CdCl^, namely 70*59 per cent., and calculating
elements for 100, we get —
„ . ., 1. Sphingomyelin ffives —
For Amidomyelin- 4oH,o4N;P09 + H,0.
Percents.
-rAt. Wts.
Atoms.
At. Wts.
Percents.
Atoms.
c
62-15
. 5-179
44
528
68-37
51-86
624
H
10-93
10-93
92
92
11-29
107-52
106
N
3-37
0-24
2
28
2-96
2-
28
P
3-65
0-117
1
31
3-24
1-
31
0
19-90
1-24
10
160
17-14
10-2
160
839
949
Diagnosis and Separation of Amidomyelin from Sphingomyelin.
Elementary analysis, with special regard to the relations be-
tween N, P, and C, is as yet the only means of establishing a
diagnosis between these two principles. It is satisfactory that in
the first steps of brain extraction they separate in the main ;
amidomyelin remains with paramyelin and lecithin, while sphingo-
myelin remains with the cerebrosides and cerebrinacides. But
the analyses of sphingomyelin in early stages of purification make
it probable that it is mixed with a mononitrogenised phosphatide
as insoluble in cold spirit as itself, a body closely resembling or
identical with paramyelin. From paramyelin amidomyelin is
easily separated by the benzol process aj^plied to cadmium
chloride salts. This process will therefore aid to separate para-
myelin from sphingomyelin also. But it has not yet been possible
to exactly fix the bearing of sphingomyelin and its cadmium
chloride salt towards benzol, nor to establish its absolute diagnosis
and absolute separation from amidomyelin when both occur in a
state of admixture with each other. Neither has there been time
or material for the study of the chemical constitution of amido-
myelin by means of the chemolytic method, which I have shown
104 CHEMICAL CONSTITUTION OF THE BE A IN.
abundantly in the course of these researches to be the only means
for obtaining jirecise final knowledge regarding the atomic com-
position and weight of these marvellous ingredients of the brain,
nerves, and protoplastic centres.
2. Amidokephalin, its Isolation, Analysis and Compounds.
Amidohephalin. — The following preparation was made from a
specimen of crude kephalin which had not undergone the purifying
process w^ith water, filtration, and hydrochloric acid. It had been
thrice precipitated from ether by alcohol, and when last dissolved
in ether, had stood during twenty-four hours in ice, to deposit
traces of myelin and sphingomyelin.
Summary of Analyses of the Free Kephalin emjjloyed in this
Experiment.
-^by At. Wgts.
P as 1.
P = 3.
c
59-51
4-96
42
127
H
9'31
9-31
79
238
N
2-73
0-195
1-6
4-98
P
3-64
0-117
1
3
0
24-81
1-550
13
39-7
100-00
It will thus be seen that all the elements are in proportions
required by pure kephalin, except the nitrogen, which is six-tenths
of an atom too high, or amounts to nearly five atoms, if three
atoms of phosphorus are assumed.
If, as is probable from analogy v/ith amido- and sphingomyelin,
there are kephalins containing amidated acids, as proximate con-
jugated compounds, which amidated acids have also now and
then been found in the chemolyses of kephalin, then it might be
supposed that out of six fatty acid radicles in three molecles of
kephalin, two fatty acid radicles were amidated.
The facts are expressed by the formula —
t'«H,„K,PO„ )
The preparation was jjroved to be free from sulphur by special
analysis. Some anomalies in details evidently do not yield to
hypothesis.
niOS.PHORmED FEIN OIF LES OR FHOSFHATIDES. 105
Transformation of this Freparoiion into Lead Salt.
The specimen of kephalin was therefore again frozen in ether,
and deposited a vestige of white matter ; the clear sohition was
then poured into absolute alcohol containing lead acetate ; the
precipitate was filtered, washed, suspended in absolute alcohol,
warmed to 45° to 50°, filtered hot, to extract soluble matters.
No deposit occurred in this alcohol on cooling. The lead salt
was dried in vacuo, powdered, and found highly electric. It was
suspended in and extracted with ether.
Summarij of Analyses:
C 44-U
H 6-583
N 1-07
Pb 27-86 27-49 26-10 26-54 Mean - 26-99
P 2-97 3-09 Mean== 3-03
0 18-25
100-00
Calculation shows that in this compound the lead does not
stand in any stoichiometric relations to any one element. It is in
excess by about one-fourth over the quantity it should be if one
atom of kephalin were combined with one atom of lead. De-
ducting the lead, and calculating the organic matter, i.e. 73-01 as
100, we get —
K in Pb salt. Free K before. Pure K gives. Theory requires.
C 60-5 59-51 60-00 60-28
H 9-1 9-31 9-39 9-44
N 1-46 2-73 1-68 1-67
P 4-06 3-64 4 27 3-70
0 25-02 24-31 24-66 24-88
100 00 100-00
The ether and lead treatment, the combination and purification
thereby eff'ected, have therefore brought this kephalin much nearer
to the composition of the purest.
3; Sphingomyelin. — Type of the Dmiidated Phosphatides,
WHICH contain no GLYCEROL : ITS ISOLATION, ANALYSIS,
Chemolysis, and Compounds.
In chemical researches which have to deal with the separation
of the ingredients of complex mixtures, the difficulties generally
rise with the atomic weights of the bodies to be treated. Of
106 CHEMICAL CONSTITUTION OF THE BRAIN.
this rule, S2)hingomyelin, a body the atomic weight of which is
probably one of the highest after those of the albuminous class,
has furnished a striking confirmation.
Sphingomyelin is the principal, but not the only, phosphorised
ingredient of the so-called cerebrin mixture, 'which remains when
white matter is exhausted by ether. Out of this mixture there
have been isolated entire series of immediate principles ; first and
best studied, the cerehrosides, with phrenosin as their chief ;
secondly, the cerehrlnacides, which contain more oxygen than the
former, and combine with lead ; thirdly, bodies containing sulphur
as an essential ingredient, hence termed cerehrosnlphatides. To
these we have now to add the description oi peculiar ijliosphatides,
and certain nitrogenised substances free from phosphorus, which
may be termed nitrogenised fats or amidolipotides. The processes
described in the following are those which actually led to the
discovery by which they were rewarded. But it is probable that,
after a complete knowledge of the properties of the newly dis-
covered bodies has been obtained, these processes may be much
simplified and shortened.
Process of separating apparently homogeneous Crystallised Bodies from
the Alcohol used for the separation of the Cerebrosides ; which
Bodies will he shown to he Mixtures hy Reagents.
After the removal of phrenosin and kerasin by crystallisation
from absolute alcohol frequently repeated, there remained alco-
holic solutions, which made no further deposit on standing. These
were distilled to a small bulk, and deposited a white crystallised
matter. (An attempt to separate anything out of the matter by
benzol proved abortive ; cold benzol extracted but little, on
application of heat all dissolved, and on cooling the mixture set
into an unmanageable jelly. The benzol was therefore distilled
off, and the matter treated anew with spirit.) The crystalline
matter was dissolved in hot spirit of 85 per cent, strength, and
an alcoholic solution of lead acetate was added, after this a slight
excess of ammonia. The cerebrinacides contained in the body
were thus precipitated as lead salts. From these the solution
was separated hot by filtration. After cooling, the alkaline
mother-liquor was separated from the crystalline deposit. The
washed crystalline mass was dissolved in hot spirit, and left
much matter in a fused state (second lead stearoconote). A part
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 107
of the matter being yet insoluble lead compound should remain
undissolved ; but when fused it encloses a quantity of soluble
matter, which then remains inaccessible to the spirit. It has
been found usefid to emulge the crystalline matter with some
water before heating it with spirit. This resolution in hot spirit
and recrystallisation are repeated four times, or until no lead salt
remains insoluble, and until the product is uniformly white, and
crystallised in stars and rosettes of needles, clearly visible under
the microscope. It is dried in vacuo.
Properties of the Product.— With water it forms a permanent
jelly or paste like starch pap. With sugar and sulphuric acid it
gives an immediate purple reaction ; with sulphuric acid alone it
becomes thoroughly purple. Heated to between 90° and 100° in a
water-oven, it becomes a little soft and a little coloured ; when
cold, it becomes again pulverisable. It fuses at about 150°,
assuming a brown colour. It yields to absolute ether-alcohol a
considerable amount of matter, which crystallises in the original
rosettes and clichotomically branched masses, like lycopodium.
Preliminarij Quantations of Elements. — Synopsis of Results.
Found
in 100.
-^At. Wgt.
-^P = l.
C
58-81
4-900
68-05
H
11 55
11-55.
160-40
N
2-63
0-188
2-61
P
2-24
0-072
1-
0
24-77
1-548
21-50
These data show that the body, though crystallised as described,
was yet a mixture. Its principal ingredients were sphingomyelin,
in which P : N = 1 : 2, and Jcerasin, which latter mainly explains
the excess of carbon and nitrogen and attached water, as will be
shown below.
Comparison with this Rosette or LycopocUum-Uke Body of a similar
Body obtained from the Cerebrin Mixture by Ether, together lulth
the Kephalin, etc.
The ether extracts from white matter containing all kephalin,
myelin, cholesterin, etc., were concentrated and mixed with an
alcoholic solution of lead acetate. Kephalin and myelin became
insoluble as lead salts. When the precipitated matters were
boiled with spirit,^nd the filtrate wa?. allowed to cool, cholesterin,
together with a lead compound and the new body were deposited.
108 CHEMICAL CONSTITUTION OF THE BRAIN.
The deposit was isolated, dried, and extracted with ether not in
excess. Cholesterin dissolved, while the lead salt and new body
remained undissolved. The latter mixture was treated with
boiling alcohol, which dissolved the new body, and deposited it
on cooling in crystalline rosettes, while the lead salt remained
undissolved as a fused mass. By frequent recrystallisation of
the new body as long as it left any fused lead-compound, it was
at last obtained quite free from lead and white.
Chemical and Physical Frojjerfies. — White crystalline mass.
Becomes soft at 90°, without loss of water. Gives Kaspail's
reaction with and without sugar, from which the presence of a
cerebroside may be inferred. The lead compound gave the Easpail
reaction with and without sugar, if at all, very indistinctly.
Analyses of the second Rosette Body. — Synopsis of Results.
Percents.
-^At. Wgt.
^P = l.
c
58-67
4-889
119
H
1M8
11-180
272
N
2-78
0-198
4-82
P
1-33
0-041
1
0
26-04
1-627
39-5
The crystallised body just analysed is a mixture of a dinitro-
genised phosphatide, sphingomyelin, with a cerebroside, as was
clearly shown by the application of reagents. But the nature of
the cerebroside in this case was not ascertained as in the former
case.
Isolation of Spjliiagcnnyelin by Cadmium Chloride Process. — Many
attempts (of which only two have been described in the fore-
going) having been made to isolate a constant product by mere
crj^stallisation with solvents, without success, the cadmium chloride
process was again adopted. The reagent was mainly applied to
cold alcoholic solutions, and had the following effect. A pre-
cij^itate of CdCl^ salt ensued immediately. If this was filtered
off quickly, a second more gelatinous precipitate fell, consisting of
almost pure kerasin. The CdCl^ salt also contained yet some
kerasin. From this it was separated by boiling spirit ; the CdCl^
salt was deposited mainly above 28°, the kerasin entirely below
28°, and on long standing provided that the amount of kerasin
did not rise above 1 part in 321 parts of spirit. Of the CdCL
salt, spirit retains less than a half per cent, (weight in volume) in
solution. It is probable that the body or bodies which combine
PHOSPHOFJSED PRINCIPLES OR PHOSPHATIDES. 109
with CdCl^ (sphingomyelin and other phosphatides) on the one
hand, and kerasin on the other hand, keep each other in solution
by some attraction which they have for each other, in which
sphingomyelin acts as base, kerasin as acid, the result being a
kind of salt which is more soluble in spirit than each of its com-
ponents by itself. The alcoholic solution from which the CdCl^
salt and kerasin have been precipitated, must be allowed to stand
long, and be repeatedly concentrated, to remove all kerasin. It
then yields a precipitate with platinum chloride and hydrochloric
acid, which is a compound of a phosphorised and nitrogenised
body, assurin, with the reagents employed. The mother-liquor
yields Icrinosin and hregenin, nitrogenised matters free from phos-
phorus, and belonging to the new class of amidated lipotides or
nitrogenised fats, as will be fully described lower down.
Gradual Purification of the CdCl.2 Salt of SjMngomijelin by recrystaU
lisation from boiling Spirit and Extraction with boiling Ether.
In the course of these processes it was found that the pre-
cipitates might contain varying quantities of CdCl2, and that
these niight correspond to several compounds. Sphingomyelin
might combine vrith one or two molecles of CdCl^ ; the second
phosphatide might combine with one molecle of CdCl^ only, being
a mononitrogenised body, probably paramyelin. The CdCl2 might
also be depressed by the admixture of kerasin. It was also found
that the CdCl., would shift, so as to increase in one fraction, while
diminishing in another of a previously unitary preparation.
The changes of these salts in general, and the extraction of the
kerasins and other admixtures in particular, were followed analy-
tically. Thus a CdCl salt gave 5-97 per cent. CI and 10-89 per
cent. Cd= 16-86 per cent. CdCl^. The 5-97 per cent. CI require in
theory 9*41 per cent. Cd = 15-38 per cent. CdCl^. The salt here
obtained was the one with one molecle of CdCl^ ; sphingomyelin
with 51 carbon atoms and CdCl2 postulates 164 per cent. CdCU ;
the body with 53 carbon atoms requires 15-98 per cent. CdCl2.
Another salt from ox cerebrosides mother-liquor of recrystallisa-
tion was a mixture of sphingomyelin CdCl^ in which N : P = 2 : 1,
and of a phosphatide CdCl2 salt, in which N : P = 1 : 1, probably
paramyelin. The salts were crystallised from hot spirit above
30°, and recrystallised until free from kerasin.
They contained 13-14 per cent. Cd, 3*24 per cent. P, and
no CHEMICAL CONSTITUTION OF THE BRAIN.
9-12 per cent. CI, 2-28 per cent. N, 50-70 per cent. C, and 9-08
per cent. H.
The chlorine found in analysis (1) postulates 14* 3 per cent. Cd.
There is, therefore, a slight deficiency of this element. The
organic molecle has therefore to be calculated by deducting the
latter larger figure for Cd, namely, 14 '3 per cent., thus :
CdCl^ - - - 23-42 per cent.
Organic molecle - 76*58 „
100-00
This quantity of CdCl^ indicates that the mixture contains
sphingomyelin monocadmium chloride with sphingomyelin di-
cadmium chloride, bodies which will be discussed more explicitly
lower down. The nitrogen is to the cadmium chloride in such a
proportion that the basicity of sphingomyelin is unsatisfied to
the extent of about one quarter, or upon two atoms of nitrogen
there is very nearly one and a half molecle of cadmium chloride
(1-48).
Synopsis of the foregoing Analytical Results.
Per cents. -^-At. Wgts.
50-70
9-08
2- 28 0-162
3- 24 0-104
11-28
I 23-42
100-00
c
H
N
P
0
Cd
CI,
Calculation of Elements in 100 of Organic Molecle.
Percents.
H-At. Wgts.
c
66-21
5-5175
51-8
H
11-86
11-860
111-2
N
2-98
0-213
2-
P
4-23
0-136
1-26
0
14-73
0-921
8-64
The relations of C : X are the same as those of the same elements
in the crystallised free body, which will be described further on.
Consequently the phosi)horus is somewhat too high.
PHOSPHOPilSED PRINCIPLES OR PHOSPHATIDES. Ill
Further Purification of the Conipound bj Ether, and by recrydallisa-
tion. — The mixture was now subjected to a process of purification
by being exhausted in a closed apparatus with boiling ether.
AVhen new ether failed to extract anything during ah entire day
of boiling, the compounds were recrystallised from spirit, again
dried, and again exhausted with ether. If now fresh ether ex-
tracted nothing, the salts were considered to be fully exhausted.
(Some krinosin and bregenin were extracted.) In this manner a
number of prej)arations were purified, with the result that in all
the carbon sank a little, while cadmium chloride rose in quantity,
but nitrogen remained to phosphorus as 2 : 1*28, as will be seen
from the following analyses.
Synojms of Quantations and Theory of Salt.
Percents. -j-At. Wgts.
C 46-11 3-84
H 8-66 8-66
N 2-24 0-160
P 3-22 0-104
0 14-37
Elements and Theory of Organic Matter.
Percents. -^At. Wgts. -^N = 2
C 61-80 5-15 48-5
H 11-60 11-60
N 3-00 0-214 . 2-
P 4-31 0139 1-28
0 19-29 1-205
We have therefore here also the phosphorus too high for sphin-
gomyelin, namely, N : P = 3 : 2, from which it may be surmised
that the admixture was partially a mononitrogenised phosphatide
(paramyelin ?), partly a diphosphatide such as will be described
further on.
As no even relation between nitrogen and phosphorus could be
obtained by any kind of treatment, particularly frequent recrys-
tallisation, to which the compound was subjected, it was deemed
necessary to decompose it and study the free body. It was
evident from the relation C:N = 51-8:2 that the organic body
was not a phosphatide of the lecithin group, in which C :N = 42 : 1,
112 CHEMICAL CONSTITUTION OF THE BRAIN
or thereabouts, while the amount of nitrogen pointed to the pro-
babihty that the body was constituted simihirly to the amido-
myelins which had been hypothetically assumed to exist in
analysed CdCl., and PtCl^ compounds. In fact, it was at once
perceived to present the proportions between C and N which had
been observed upon apomyelin described in my first research on the
brain. But as it had been left doubtful whether this apomyelin
was not a product rather than an educt, great care was taken
to prevent any chemolytic influence from acting on the body, so
that the doubt just mentioned might be eliminated. The crys-
tallisation of the free substance showed at once the presence of
fico imnciplcs, of which the first, sphingomyelin, was obtained pure
by crystallisation, while the second one was obtained pure by
more circumstantial processes to be described later.
PrepardtioR of pure Sphingoniijeliii from its Cadmium Chloride
Compound.
Removal of the Cadmium by Diffusion and Dialysis. — AYhen the
compound is placed in Avater, this will extract much of the CdCl.,,
but not all, by dissociation and diffusion. But the liberated
sphingomyelin is in the state of colloidation, which increases with
the length of contact with water until it reaches its maximum.
The liquid can thus no longer be filtered from the sphingomyelin,
as the latter immediately obstructs the pores of the filter.
Dialysis may be resorted to, the apparatus being arranged with
plaited vegetable parchment dialysers as described on p. 101.
AVhen the dialysate is free from CdCl^, the sphingomyelin and
water paste must be concentrated, dissolved in hot spirit, treated
with hydrothion to remove the last portions of cadmium, and
set to crystallise.
The sphingomyelin which was examined in the first four
analyses to be related below was prepared with the aid of
dialysis, as follows : 40 g. CdCl., salt was boiled with 1 litre spirit ;
23 g. dissolved, while 13 g. remained insoluble in the quantity of
spirit mentioned. The hot spirit solution deposited a quantity
of salt above 30^ and was filtered at 30°. The salt thus obtained
was subjected to dialysis, and washed with water until free
from chlorine. It was then recrystallised from spirit, and dried
at 100".
The liquid which had deposited the CdCU salt above 30° was
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 113
evaporated, and the deposit filtered off. It was also decomposed
with water, and the body free from CdCl2 was analysed. It
yielded results which have been recorded separately. If my
object were solely that of a mere chemist in search of new pure
compounds, I should have thrown this and other mother-liquors
away. But the object of physiological and pathological research is
not to find some principles which will pay the chemical operator,
but to find all the principles which may be contained in an organic
physiological or pathological mixture.
The following process yields sphingomyelin in a shorter time
than dialysis. The purified salt, free from kerasin and all matters
which ether can extract, is susj)ended in spirit, and treated with
hvdrothion to saturation. The fiask containinsr the mixture is
now placed in a water-bath, the water of which is gradually
raised in temperature until the spirit in the flask boils. The in-
fluence of the sulphuretted gas is continued until a filtered sample
of the spirit solution is not changed by the hydrothion any more.
The mixture is now filtered through paper in a funnel kept hot
by a steam-jacket. The filtrate is left to crystallise. It yields
sphingomyelin, etc., partly as hydrochlorate, which are collected
on a filter, washed with spirit, pressed between bibulous paper,
and dried in vacuo over oil of vitriol. The neutral and dry salts
are now again dissolved in warm spirit, and to the solution is
added gradually, and in small quantities, as much finely pul-
verised mercuramin as may be necessary to bind and retain in the
precipitate all the hydrochloric acid combined with the phos-
phatides. When an excess of mercuramin is present — i.e.^ when
the deep canary-yellow colour of the mercuramin is no longer
changed to white, but remains as a yellow deposit at the bottom
of the flask, distinct from the white mercuramin hydrochlorate —
then the organic principles are free from hydrochloric acid ; the
filtered solution, on cooling, deposits first sphingomyelin, to be
purified by recrystallisation ; the concentrated mother-liquor de-
posits the mononitrogenised companion mainly. (Without the
mercuramin treatment the sphingom/elin may retain from 0 8 to
1-32 per cent. HCl.)
Physical and Chemical Properties of Sphingomyelin. — Sphingo-
myelin is easily soluble in hot spirit or absolute alcohol, and
crystallises therefrom in dense white masses, needles, stars, and
hexagonal j)lates. It does not become waxy after drying, but
^8
114 . CHEMICAL CONSTITUTION OF THE BRAIN.
retains a pulverulent dryness. It is little soluble in cold absolute
alcohol, but can be separated thereby from phrenosin, which is
less soluble, and from lecithin, which is more soluble. It is
almost insoluble in ether, even when some hydrochloric acid has
been added. It cannot be separated from kerasin, to which it
stands in the relation of a base, by recrystallisation alone. The
intervention of cadmium chloride is necessary to effect this
sej^aration. The compound of sphingomyelin with cadmium
chloride is less soluble in spirit than the pure sphingomyelin, and
is deposited in a much shorter time and at higher temperatures
than kerasin ; for the sphingomyelin CdCl2 falls above 28°,
kerasin (in solutions which contain less than 1 g. in 321 cc. of
spirit of 84 per cent.) below 28°, on standing. Sphingomyelin
swells in water, and becomes distributed in it so as to form a
turbid semisolution or emulsion. In the course of dialysis it
sometimes contracts and sinks in the w^ater. Its compounds,
particularly those with CdClg, become decomposed under the in-
fluence of water ; sphingomyelin assumes the colloid form, and
the combined metals or salts pass into solution in the water. On
this property rests the process for liberating sphingomyelin from
crystalloids by dialysis.
Quantaiions of Elements in Sphingomyelin from Ox Brain.
Synopsis of Analyses and Theory,
Percents.
-hAt. Wgts.
c
65-37
5-445
51-86
H
11-29
11-29
107-52
2-96
0-211
9 .
P
3-24
0-105
1-
0
17-14
1-070
10-2
The probable formula is C^^H^q^NoPOc) + H^O.
Comparison of Sphingomyelin witli Aponiyelin from Human
Brain.
In 'Eeports,' No. III. (1874), p. 164, 1 have given the analysis of
a specimen of a kind of myelin from the human brain which I
termed Aponiyelin, and which had been obtained from a platinic
chloride compound by recrystallisation from boiling alcohol, and
decomposition of the product by hydrothion. The following
figures were given :
FHOSPHOFJSED PRINCIPLES OR PHOSPHATIDES. 115
A])omyeliii, Man.
Percents.
—At. Wgts.
-f-P =
c
67-01
5-5841
54
H
11-35
11-35
109
N
3-00
-2142
2
P
3-23
•1041
1
0
14-694
•9155
9
CI
0-761
This was, therefore, clearly a sphingomyelin with 54 C ; like
the sphingomyelin from the CdCl^ compound, it retained a small
quantity of hydrochloric acid, which was estimated in the analyses
and recorded as CL A sphingomyelin prepared from CdCl^ salt
by H2S, was found to retain 1-32 per cent. HCl, which is only
about a third of the theoretical amount required by a simple
hydrochlorate. In both cases the hydrochlorate was probably
decomposed by the influence of watery solvents.
Chemolyses of Siohingomyelm, ivith a view of ascertaining its
Chemical Constitution.
I have made four distinct experiments on this subject ; bat as
they are not completed, I am only able to state the salient results
of the operations as far as they go.
Experiment I. — Six g. sphingomyelin were mixed with 1 2 g.
barita hydrate and heated to 105° during five hours. There were
obtained a little sjjhingosin (precipitated from the alcoholic extract
by sulphuric acid, soluble in excess), an acid which differed from
sphingomyelin by the absence of the group of neurin, sphingomyelic
acid, probably (if the sphingomyelin employed had the formula
^53^106-^2-^012) li^ving the composition C4gHc)5NPOj2- In this
acid, it will be observed, P:N=1:1. There was further ob-
tained a barium salt soluble in ether, and an alcohol sphingol, the
two latter bodies being products of the continued decomposition
of a portion of sphingomyelic acid.
Experiment II. — Twelve g. sphingomyelin, 12 g. barita and 50 g.
water were heated to 100° for ten hours. There were obtained
1 -1 g. of neurin as platinic chloride salt, while theory requires
1*3 g. from 12 g. sphingomyelin. No trace of glycerophosplioric acid
was observed. There was a barium salt insoluble in alcohol and
ether, which contained P:N = 1:1-02 (P-2-75 per cent.;
N='l-27 per cent.) sphingomy elate of barium. No sphingosin
116 CHEMICAL CONSTITUTION OF THE BRAIN
was obtained. But the new alcohol, sphingoid was isolated in suffi-
cient quantit}^ to be analysed.
Anahjsis of Sphingoid a new Alcohol, from Sphingoniyelin hj
Chemolysis tuith Barita.
Dried at 98° in a platinum boat.
Synopsis of Analyses and Theory.
Percents. -^-At Wgts. -i-byO = l.
(1) C 76-30 6-37 9-07
H 12-78 12-78 18 7
0 10-92 0-68 1-
(2) C 76-34 6-361 9-08
H 12-45 12-45 17 92
0 11-21 0-7 1-
The new alcohol has therefore the composition expressed by
either CgHjgO, or by C^gHggO^. In the latter case it would be the
third stearic isomer.
Analysis of the Neurin-Platinum-Chloride Hydrochlorate ohtdined
from this Chemolysis.
The salt CioH26N,02, 2HCl,PtCl^, of atomic weight 618-4,
Requires in 100. Found in Salt.
Pt 31-921 31-79
CI 34-44 34-64
Experiment HI. — Twenty g. sphingomyelin and 40 g. barita, with
500 g. of water, were heated to 135° during twelve hours. There
were obtained neurin ; further a second alkaloid precipitated by
sulphuric acid from its solution in absolute alcohol, yielding as
sulphate by analysis the formula 2(C2oH4jN02)H2SO^, being
possibly somewhat impure sphingosin ; sphingol ; a fatty acid,
which as barium salt and as lead salt was insoluble in alcohol and
in ether, in the free state had the composition expressed by the
formula C^gH^^O^, crystallised, and fused at 57°, therefore almost
as much below ordinary stearic acid, which fuses at 69-5°, as
neurostearic acid fuses above this, namely, at 84°. The acid is
therefore the fourth isomer of stearic acid, the third discovered
in these researches, and is named sphingostearic acid.
No glycerophosphoric acid was ol)tained, but the insoluble
barium salt contained much harium plioxphate^ which was isolated
PHOSPHOIUSED PFilNCIPLES OR PHOSPHATIDES. 117
the phosphoric acid was combined with molybdate of ammonium,
and fully identified.
Experiment IV. — Twenty-five g. sphingomyelin, 50 g. haiita,
and some water were heated in the autoclave to 105°— 120° for
seventeen hours. There were again obtained neurin, sphingosin,
sphingol, barium salt insoluble in ether, and a neutral body mixed
with sj)hingol ; this latter mixture or compound had the formula
Cg^HgiNO^, with 2-18 K
Some intermediate, in quantity subordinate, products, such as
a barium salt soluble in ether and precipitable by alcohol, remain
for further study.
Theoretical Results of these Chemolyses.
The conclusions to be derived from these chemolyses bearing
upon the chemical constitution of sphingomyelin are of the utmost
interest and importance.
In the first place, no glycerophosphoric acid was obtained
in any one of the chemolyses. If I recall the many experiments
•made regarding the constitution of other phosphatides, such as of
kephalin or of lecithin, and the relative facility with which gly-
cerophosphoric acid was isolated as barium salt and identified as
calcium salt, I cannot suppose for a moment that glycerophospho-
ric acid has been present and escaped observation. I therefore
came to the conclusion that the glycerol found in a portion of the
phosphatides was not, as has been hitherto supposed, essential to
the composition of those bodies. From this moment it was also
evident that glycerol could not, or need .not, be the basal radicle
of these compounds, not even of those which contained it, and
that therefore the phosphorised brain educts could no longer be
considered as glycerides, or ethers of the alcohol glycerol, but had
to be differently interpreted. The only feature common and
essential to all the phosphorised educts, and the one from which
in consequence they derived their appellation, was that they con-
tained phosphorus, which by ordinary analytical processes re-
mained in the form of phosphoric acid. There was no reason to
suppose that the phosphorus was present in the organic principles
in any other form than that of phosphoryl, the radicle of phos-
phoric acid. (When they were chemolysed they took up water,
and yielded hydrated radicles, such as have been described,
acids, alkaloids, or organic bases, and alcohols.) Consequently I
118 CHEMICAL CONSTITUTION OF THE BRAIN.
assumed that the ha sal radicle of all phosphatides was that of phosphoric
acid ; that in this acid one, two, or three molecles of hydrox)jl might be
replaced hj radicles of alcohols, acids, or ba.ses, and that to a moled e
formed by three such substittdions there might yet be attached, by substi-
tntion of an element in a radicle, itself already substituted (side -chain)
m- by addition as anhyd.ride, a fourth radicle, and that therefore
sphingomyelin might be constituted according to the following
formulae :
fHO
Phosphoric acid = OP J HO
I HO
f Acid radicle.
I Alcohol radicle.
Sphingomyelin = OP -{ Base radicle (substituted).
I Base radicle (attached as side- chain or
[ added as anhydride).
I repeat the formula, substituting elementary formulae for the
functional symbols :
fC.sH^A 1
[ C,H,,N J
To this formula we are obliged, by the results of the analyses
of the three sphingomyelins, to add at least 2H^0, perhaps 3H^0,
and by this operation we obtain a total formula of C^gHjoiNoPOj^,.
But this last formula is only a case out of many possible
formulae. It is clear that if the molecular formula of sphingol
were only C.^H^gO, the formula above given would either have to
be lowered to C^,^, etc., or the sphingol would have to be assumed
to be doubly rej^resented. Neither hypothesis enjoys the advant-
age of probability. I therefore do not presume to fix the exact
formula of sphingomyelin, particularly as I have shown that there
are various sphingomyelins (one being apomyelin), but I maintain
that this research has established as a type of brain-educts sphin-
gomyelin, which is a phosphatide, and contains an alcohol not
being glycerol, an acid, and two nitrogenised radi'jles as proximate
constituents.
Coinpounds of Sphingomyelin. — The body combines with hydro-
chloric acid, and this compound is more soluble in spirit than the
free body. The hydrochlorate is easily decomposed by watery
solvents, so that while a monohydrochlorate should contain 3 '76
PHOSPHORISED PRINCIPLES Oil PHOSPHATIDES. 119
per cent. HCI, there were found in a hydrochlorate recrystallised
from spirit ow\y 1 -32 per cent. HCI.
Compounds of Sphingomyelin ivitli Caclmium Chloride. — The vary-
ing quantities of cadmium chloride in such compounds have been
a source of great trouble and doubt, which could only be solved
by countless preparations and analyses piloted by hypotheses
and the atomic theory. Thus a salt was found to contain
16-86 per cent. CdCl^, and to correspond to the formula
CjjHggN^POiQCdClg, which requires 164 per cent. CdCl^.
Another salt from the human brain contained 26-59 per cent.
CdClg, and was therefore supposed to be a not quite saturated
compound approaching that with two molecles of CdClg. For the
Q.^-^ sphingomyelin v;ith 2CdCl2 requires 28 per cent. CdCl^ ; the
formula with C^^ requires 2 7 '9 per cent. CdCl^.
Sphingomyelin is therefore, like amidomyelin, a dipolar alkaloid,
and capable of fixing one molecle of CdCl^ to each of its two
nitrogen radicles.
Consequently any sphingomyelin and CdCl^ compound which
contains quantities of CdCl2 intermediate between 16-4 per cent,
and 28 per cent, is a mixture of the monocadmium chloride, with
the dicadmium chloride compound. Such a compound must of
course contain P : N = l : 2. Where these relations do not subsist,
but where the relations of P : N approach more or less those of
2 : 3, the cadmium chloride may be diminished by the presence of
a compound with it of a phosphatide such as paramyelin, in which
P : N = 1 : 1. It will therefore be seen that the higher the amount
of CdCl.2 found in a new precipitate, the more likely is the body
to contain sphingomyelin (or amidomyelin). If the CdCl^ sinks
much towards 20 per cent, or below, the precipitate or crystallisa-
tion either contains much of the monocadmium chloride compound,
or contains a mononitrogenised body, which maybe either a body
analogous to sphingomyelin, but containing one nitrogenised
radicle only, or a lower phosphatide such as lecithin or paramyelin.
We have therefore, with regard to sphingomyelin, the possibility
of the existence of two compounds and an indefinite number of
their mixtures in diff'erent proportions
C5.H99N,POio-FCdCl,, At. W. = 1113, contains 16-4 per cent.
CdCl^.
C5iH99N2POio-f 2(CdCl2), At W.-.1296, contains 28 per cent.
120 CHEMICAL CONSTITUTION OF THE BRAIN.
If there are, as is probable, many sphingomyelins, then each
variety would have its several CdCl., compounds.
All these CdCl2 salts are beautifully crystallised and white.
When they are recrj^stallised from spirit they lose solubility as
the extraction proceeds. These changes are particularly observed
upon salts which are not fully saturated with CdCl., : the fully
saturated salts behave in a regular and stable' manner. It is
therefore necessary in the first crystallisation to offer to sphin-
gomyelin an excess of cadmium chloride, and to test the mother-
liquors of recrystaUisation from time to time to see that they do
not contain unsaturated more soluble salt or free sphingomyelin
in solution. Spirit of 85 per cent, strength, after having been
saturated boiling with the dicadmium chloride salt and allowed
to deposit all it can during twenty-four hours, will keep in solu-
tion half a gramme of the salt in 100 cubic centimetres.
C. SUBGROUP OF DINITROGENISED DIPHOSPHATIDES.
N : P = 2 : 2.
ASSURIN : ITS ISOLATION AND ANALYSIS.
This body is found in the alcohol extracts of the cerebrin
mixtures after sphingomyelin and kerasin have been removed
from them in the manner above indicated. When to such a
solution platinum chloride, acidified with some hydrochloric acid,
is added, a precipitate ensues which is insoluble on boiling. In
solution there remains a body, which I have termed Isfarin, and
which does apparently not combine with platinum chloride.
Both bodies, assurin and istarin, seem to have some attraction for
each other, like sphingomyelin and kerasin, which causes them to
crystallise together, so as to represent a uniform appearance of
star-shaped masses of crystals. This union is apparently never
definite, but the projDortions of the ingredients shift according to
the mass, concentration, and temperature of the solvents used for
their extraction. On the whole, frequent recrystaUisation from
.spirit causes the phosphorus in the mixture to rise, and the nitro-
gen to sink relatively to the phosphorus. But the principle here
to be described is isolated as yet only by platinum chloride.
Assurin Hydrocldoraie Flatiiium Chloride.
Yellow crystalline powder, insoluble in boiling spirit, and in
ether.
PHOSJPHORISED FRINCIPLES OR PHOSPHATIDES. 121
Synopsis of the Results of the First Series of Analyses and Theory.
Percents.
^At. Wgts.
^Pt=-1.
Organ. MolecL
c
49-25
4-104
91-20
45-6
H
'8-74
8-740
194-22
97-0
2-50
0-178
3-95
1-97
P
6-52
0-210
4-66
2-33
0
13-69
0-855
19-00
9-5
Pt
9-03
0-045
1-00
CI
10-27
0-289
6-42
These data lead to a formula 2(C,^H9^N,P209HCI)PtCl,.
Synojjsis of the Results of the Second Series of Analyses and Theory.
Percents.
-f At. Wgts.
-f Pt = l.
Organ. Molecle.
c
49-01
4-084
49-8
94-3
47-
H
8-85
8-85
203-
101-
N
2-30
0-169
2-
3-77
1-88
P
6-06
0-1954
2-2
4-49
2-24
0
15-66
0-98
22*
11-
Pt
8-57
0-0435
1-
CI
9-55
0 269
6-18
These data lead to a formula 2(C^J4ioiN2PoOn^iCl)PtCl^,
which differs a little from the former one, but phosphorus remains
slightly exceeding, nitrogen below the theory derived from the
p)latinum chloride as starting base. Assuming nitrogen at two
atoms, we come to nearly 50 C, but encounter again an excess of
10 per cent, in the phosphorus. But the great features of the
results of the quantations are evident. We have to deal with a
phosphatide in which the radicle of phosphoric acid is contained
twice, and which we may therefore term a dii:)hosphatide. In this
principle there are contained two atoms of nitrogen, which, from
analogy with other phosphorised bodies, we may suppose to be
contained in two different nitrogenised radicles. But even if the
two atoms of nitrogen were contained in one and the same radicle,
it would still be perfectly correct to term the principle a dinitro-
genised diphosphatide. For the nitrogen, although somewhat
deficient in both sets of analyses, amounts nevertheless to 3*77
molecles in the latter and to 3*95 in the first set of quantations
when compared to platinum as 1.
The companion of assurin, the above-mentioned istarin, is not
X)hosphorised, but it is very difficult to prepare it free from the
last traces of phosphorus. Its chemical composition is expressed
122 . CHEMICAL CONSTITUTION OF THE BEAIN.
approximately by the formula C^QHg.,NO^ ; it therefore seems to
belong to the group of nitrogenised fats to be described below.
I have prepared and analysed many specimens which have shown
the way to the ultimate complete chemical individualisation of
the substance. But there has not been time for the carrying out
of the laborious operations which are necessary for the isolation
of the quantities required. For it must be borne in mind that
befoi^e istarin is reached in a systematic course of brain analysis
all the other substances described previously must have been
removed out of the solution, as well as the residues of the solvents
and precipitants.
D. SUBGROUP OF NITROGENISED PHOSPHATIDE-
SULPHATIDES.
Eocli/ from Group of Cerehrlnacides.
Such a principle I shall have to describe under the subgroup
of the cerebrinacides, amongst which it occurs, and from which
it has not yet been entirely isolated. Indeed, it may be ques-
tioned w^hether this cerebrosulphatide contains phosphorus as a
constituent element, or only as a constituent element of an ad-
mixture. The observation, such as it is, is too important to be
left out of sight ; and, on the other hand, nothing but a research
of great dimensions will be the means of evolving the final truth
contained in it.
E. SUBGROUP OF NONNITROGENISED MONOPHOSPHATIDES.
A body typical of this subgroup was discovered by limited
chemolysis of kephalin, as descril^ed in the chapter relating
thereto, under kephalophosphoric acid. Two other bodies of this
kind were found in a lead precipitate from buttery matter. One
was a crystallised acid, the other noncrystallised. There has not
been time for advancing the knowledge concerning them ; par-
ticularly the proof is yet wanting that they are educts, and not,
like kephalophosphoric acid, products. They are, therefore,,
registered here mainly as objects for future research.
First acid from buttery matter.
Second ncid from buttery matter.
Kephalophosphoric acid (product).
PHOSPHORISED PFJNCIPLES OR PHOSPHATIDES. 125
From the mixture of the first two acids a barium salt sohihle
in ether was obtained, reminding of the bearing of the kephahn
and sphingomyelin series, which alone, as thus far known, yield
barium salts soluble in ether.
F. COMPARATIVE CONSIDERATION OF OTHER PHOS-
PHATIDES OF THE ANIMAL BODY.
PhosjjJiatide of the Milk, Ladophosphaticlc, Casein.
According to the latest researches, casein from cows' milk has
the following percentic composition :
-f- At. Wgts. Atoms.
C 52-96 4-413 197-8
H 7-05 7-05 316-
N 15-65 1-1178 50-
S 0-716 0-0223 1-
P 0 847 0-0273 1-224
O 22-78 1-4237 63-8
It will be seen that what has often been maintained before is
here again propounded — namely, that a substance believed to be
truly albuminous contains not only sulphur, but also phosphorus,
as an essential constituent. On general grounds I think this very
23robable. Indeed, the phosphatides of the brain have some pro-
perties which are so much like those of casein that former
inquirers were led by them to the belief that the brain did
actually contain casein. As casein yields by chemolysis about
4*12 per cent, of tyrosin, and as the atomic weight of the latter
is 181, the atomic weight of casein must be at least 4393. Now
if casein contained one atom of sulphur, its atomic weight would
thereby be fixed at about 4469, which does not differ much from
the number derived from the tyrosin ; but the phosphorus leads
to a lower number — namely, 3659. Seeing, however, that phos-
phorus is analytically always found a little too high, we need not
at first sight attribute too much importance to this difference.
Seeing, on the other hand, that albumen contains at least three
atoms of sulphur, we need not despair of finding, by further
inquiry, a better ratio between the phosphorus and sulphur in
casein than that which is at present apparent.
124 CHEMICAL CONSTITUTION OF THE BRAIN.
Phosphatides of the Bile, Cholophosphatides,
It is generally assumed that the bile contains lecithin. This
^issumption is based upon the fact that ox-bile, by chemolysis
with barita, yields fatty acids and neurin. Indeed, neurin was
first discovered in the bile, and originally termed choHn. I have
made some experiments regarding this question, and come to the
result that ox-bile does not contain lecithin, but contains a phos-
phatide which, to conclude from its crystallising as platinum
chloride salt, seems to have a very complicated composition. The
formula expressing the composition of the platinum chloride com-
pound with P = 1 was Cg2Hi^^N^P03g,HCl + 2PtCl^. The fact that
bile, a secretion which serves the chemistry of digestion and
■assimilation, contains, besides its specific ingredients, bodies
which, like cholesterin, are identical with important ingredients
of the brain, or, like this cholophosphatide, are analogous to
them, shows that the biolytic or biosynthetic process which leads
to the formation of bile is much more complicated than has
liitherto been supposed. One of the principal fatty acids in the
bovine cholophosphatide is stearic.
Fhosjihafides of the Blood, Heinatophosphatides.
A phosphatide was found mixed with a preparation of hemine
crystals made according to Eollet's prescription. It was extracted
by benzol and acetic acid, and from the residue of this solution
by hot absolute alcohol. From this solution it was precipitated
as CdCl^ salt ; the white salt was recrystallised and analysed. It
led to an empirical formula, C-(3Hj,5^N.,P^Oi4 + 2CdCl^. From this
it is probable that the salt was a mixture of a mononitrogenised
with a dinitrogenised phosphatide — if a conclusion may be drawn
from its physical appearance, paramyelin and amidomyelin. It
had almost the same percentic composition as a similar prepara-
tion of a CdCl^ salt obtained from brain. We may assume that
this phosphatide came from the blood-corpuscles, like the hematin
which it accompanied, and was an element of their bioplastic
constitution. Whether it was in any way centralised, like the
phosj^hatides of cell-nuclei, cannot be stated.
From an ether extract of blood-corpuscles of the ox I obtained
a cadmium chloride precipitate, of which a portion was soluble in
cold benzol — lecithin cadmium chloride; while another portion
was insoluble in boiling benzol — amidomyelin cadmium chloride.
FHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 125
Phosphatides of Nucleolar Centres of Growth [Bioj^lasm, Cells, etc.),
Cyfophosphatides.
Aggregations of cells, whether vegetable, such as yeast, or
animal, such as sperma, pus, or liver (the latter after removal of
bloodvessels and connective-tissue), are known to contain a
peculiar phosphorised substance. As this substance was supposed
to be contained in or to constitute the nucleus, it was called
nuclein. This term would be unobjectionable if all tissue
elements yielding the substance contained nuclei. However, for
our present purpose we will consider mainly those which do
contain nuclei, and leave the others for future consideration.
The nucleolar matter has mostl}^ been obtained by subjecting
the cells to a process of artificial digestion. The undigested
(i.e., undissolved) part was supposed to be unaltered nucleolar
matter. This was now extracted with dilute caustic alkali, and
from the filtered solution the nuclein so-called was precipitated
by dilute hydrochloric acid. This was washed with alcohol and
ether, dried in vacuo, and analysed. Other authors avoided the
process of digestion, and extracted the mass of cells, such as
German yeast, with soda lye directly, and treated the filtrate as
just described. Nuclein from yeast thus obtained gave from
40-42 to 41-22 per cent, of C, 5-15 to 5-52 per cent. H, 15-31 to
15-99 per cent. N, 6*1 to 6-29 per cent. P, and 0-38 to 0-41 per
cent. S ; or — ■
Means.
C 40-81
H 5 -38 •
N 15-76
P 6-19
S 0-39
But a great number of other analyses gave only from 2-58 to
3-98 per cent. P. The nuclein from pus-corpuscles gave from
2-28 to 2*62 per cent. P, about 1-7 per cent. S, and from 14 to
15-02 per cent. N, besides 49-58 per cent. C, and 7*10 per cent. H.
Nuclein from the red blood-corpuscles from geese gave 6*04 to
7-12 per cent. P, and 0-4 per cent. S; while so-called nuclein
from the so-called nucleolar formations of the yelk gave 7-10 per
cent. P, 0-99 per cent. S, and 13-46 per cent. N.
From this it will be evident that the science of the nucleins
is only in course of development, and that there are at present
126 CHEMICAL CONSTITUTION OF THE BRAIN.
two groups of these substances known — one with from 6 to 7 per
cent. P, another Avith from 2 to 3 per cent. P. The latter is the
better known. It yields, by long boiling with water, p]ios])1wric
acid, an albuminous suhtafice soluble in water, and a peptone, and
a mixture of the three alkaloids, hy]Joxanthin, xanthin, and guanin.
From the study of the phosphatides of the brain, we can have no
difficulty in explaining such a body hypothetically to be a phos-
phatide. The body with the high amount of phosphorus we could
comprehend to be a di- or triphosphatide ; whereas the body with
the lesser amount of P would have a more simple structure.
However, our only object in this place is to direct attention to
these bodies also ; for there can be no doubt that the knowledge
of each set of organic principles will enable us to advance that of
the others.
G. INORGANIC BASES EXISTING IN THE BRAIN IN COM-
BINATION WITH PHOSPHATIDES.
The phosphatides, as they exist in brain-matter, and as isolated
therefrom, are associated with certain bases, by a power of com-
bination derived from their acid character. These bases can only
b)e removed and the principles obtained in a pure state by dis-
solving the compounds in water, and adding hj^drochloric acid,
which combines loosely with the principles on account of their
alkaloidal character, to the exclusion of the bases, which then
pass into solution as chlorides.
In order to obtain some knowledge regarding the relative
amounts and nature of the bases in combination with the phos-
phorised constituents of brain-matter, a quantity of solution
obtained by the hydrochloric acid process, above described, and
which was derived from kephalin, paramyelin, and lecithin, was
submitted to analysis.
It was evaporated to dryness, and the residue ignited in a pre-
viously weighed platinum dish. During this ignition much
chloride of ammonium was given out, and its nature distinctly
proved by condensing and analysing it.
The fused mass was dissolved in dilute hydrochloric acid, and
the solution so obtained treated with excess of ammonia. The
white gelatinous precipitate which was thrown down was scarcely
coloured black by sulphide of ammonium. The precipitate and
filtrate were submitted to qualitative and quantitative analysis.
PHOSPHOBISED PRINCIPLES OR PHOSPHATIDES. 127
The Precipitate. — The colouring-matter was evidently sulphide
of iron, but it did not amount to more than a trace. [The original
solution had been subjected in a neutral state to a current of sul-
phuretted hydrogen with the object of removing some platinum
that had been used along with the hydrochloric acid, as PtCl^, in
the precipitation of the kephalin and myelin from the aqueous
solutions. This treatment must, therefore, have removed any
copper and iron which were undoubtedly present.]
The precipitate was dissolved in dilute hydrochloric acid, and
the calcium, magnesium, and phosphoric acid estimated as
follows :
The solution was nearly neutralised by sodic carbonate, after
addition of some ferric chloride, and then an excess of pure
baritic carbonate was added ; the mixture was shaken, allowed to
stand, and then filtered.
From the concentrated filtrate the barium was removed by sul-
phuric acid, and the filtrate was then precipitated in the presence
of ammonia by oxalate of ammonium. The oxalate so obtained
was converted by intense ignition into oxide, CaO, which weighed
0-1994 g.= 0-1423 g. calcium.
The filtrate and washings from the calcium oxalate were treated
as ordinarily in cases where it is desired to estimate magnesium,
and there was obtained 0*5555 g. ^i^.^fiy, = 0*1212 g. mag-
nesium.
The precipitate which had been produced by ferric chloride
and sodium and barium carbonates was dissolved in nitric acid,
and the phosphorus estimated by the combined molybdate and
magnesium methods. This gave 0'3266 g. phosphorus = 0*7480 g.
phosphoric acid (P2^5)-
The A'mmoniacal Filtrate. — This was proved to be free from mag-
nesium and calcium. The potassium contained in it was estimated
in the usual way by means of platinic chloride, giving 11*2766 g.
of 2KCl,PtCl4 salt= 3*651 g. KCl = 1*911 g. potassium. The
mother-liquor and washings were evaporated to drj^ness, and the
platinum was removed by extracting the ignited mass with water
acidified by hydrochloric acid. The extract so obtained was
evaporated to dryness and fused ; the residue weighed 3*45 g.
(NaCl) = 1*356 g. sodium. The quantities of potassium and
sodium thus found were controlled by the analysis of a separate
part of the original solution containing their chlorides, and cal-
128 CHEMICAL CONSTITUTION OF THE BKAIN.
dilating from the residue obtained on evaporation, there should
have been found :
A total quantity of chlorides = 7 '09 g.
There was found - KCl = 3 -6 5 1
and NaCl= 3-450
or a total of = 7 '101 „
Distribution of the Bases and Phosphoric Acid. — Assuming the
calcium and magnesium to have originally existed as tribasic
phosphates, and any phosphoric acid remaining over to have been
in combination with potassium, then we have :
Ca =0-1423 g. existing as SCaO,?^,.
Mg = 0-1212 „ „ 3MgO,PA.
K =0-5663 „ „ 3K,0,P205-
K =1-3447 „ I combined directly with kephalin,
Na =1-356 „ j pararayelin, and lecithin.
P =0-3266,, „ P2O5 (0-748 g.) and distributed
between the Ca, Mg, and K.
It has been proved above that kephalin was partly in combina-
tion with calcium oxide, or lime, uncombined with any other
acid. In the present research we find that there may be in
kephalin (and myelin) even a much greater quantity of potash
and soda in direct combination than of lime. Both observations
supplement each other, the first one being qualitative only, the
present one stating the quantities of the bases relatively to each
other.
H. SPECIAL STUDY OF GLYCEROPHOSPHORIC ACID AND ITS
SALTS, AS OBTAINED EROM SOME PHOSPHATIDES OF
THE BRAIN.
Glycerophosphate of lead may be prepared from the solution of
barium glycerophosphate as obtained by the chemolysis of
phosphorised matters, by j^recipitation with any soluble salt of
lead, as, for example, the chloride or acetate. In order to entirely
remove glyccrophosphoric acid in this way from the solution, it is
necessary to concentrate and neutralise the latter from time to
timei, on account of a slight solubility of the glycerophosphate of
lead.
Glyceroi)hosphate of lead prepared synthetically is granular
and white, and remains so on drying, whereas when it is obtained
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES, 129
from kephalin it dries to a hard, brittle, slightly coloured mass,
even when chemically pure.
On ignition, the salt leaves a residue of pyrophosphate of lead,
and this offers a ready means of ascertaining the purity of the
preparation. Thus, with a specimen of the salt jDrepared as
described from kephalin, it was found that 0-911 g. left a residue
of 0-744 g. Pb^PoO^, while theory requires 0-7104 g.
Glyceroi?liosplmte of calcium (normal salt) may be prepared from
the lead salt by decomposition with hydrothion, and neutralisation
of the filtrate with calcium carbonate.
This salt is much less soluble in a hot concentrated aqueous
solution than in the same amount of water in the cold. It is
therefore best isolated by filtration of a precipitate from the
nearly boiling solution.
A quantity of the lead salt, referred to above, was converted
into calcium salt, and the solution evaporated near the boiling-
point, when a w^hite deposit of calcium glycerophosphate formed.
This, when washed and dried, was analysed. 0-3400 g., after
strong ignition with the aid of nitric acid, left a residue of
0-207 g., equal to 60 8 per cent. Ca^PgO^. Pure calcium glycero-
phosphate should leave 60-5 per cent, pyrophosphate.
Another sample of the calcium salt prepared as described gave
the following figures on analysis :
which shows the composition of this salt to be according to the
formula CgH.CaPO,.
Acid Glyccropliospliate of Calcium. — A solution of the calcium
salt neutral to test-paper becomes on heating, and at the same
time as the normal salt separates, acid to test-paper ; and if
the mother-liquor is now precipitated with alcohol, there is pro-
duced, not the normal but the acid salt. A portion precipitated
in this way by alcohol was white and granular. It was dried
at 100° C.
0-827 g. left on ignition 0-478 g. residue = 57-80 per cent. Now
the normal salt would have given, as we have seen, 60-5 per
C
H
Ca
P
O
Atoms.
2-9
7-3
1-0
10
6-1
9
130 CHEMICAL CONSTITUTION OF THE BRAIN
cent, residue. This observation led to the theory of an acid salt
of this construction, C3H^CaPOg,C3H9PO,3, giving a total formula
of C^3HJgCaP20^2 ; and it was probable that on ignition such a
salt would leave a residue of half-saturated acid pyrophosphate
H.CaP.O^, losing only CeHi.O^.
On this theory, the residue should have amounted to 56-54 per
cent. ; it did amount to 57*80 per cent.
Gli/cerophosjjhate of harium prepared from synthetically made
acid and barium carbonate was white, and behaved, as regards its
insolubility in, and consequent precipitation from hot aqueous
solution, similarly to the calcium salt. But this property of
separation of the salt on boiling the solution seemed to be only
transient, for after a solution had stood for sixteen hours after it had
been so precipitated, no precipitation occurred on again boiling the
solution, and ammonia produced a voluminous precipitate in the
solution, apparently indicating that a partial decomposition of the
salt had occurred.
Some of the salt was prepared from the solution resulting from
the decomposition of kephalin by barita, by precipitation with
alcohol. This was redissolved in water, and reprecipitated by
alcohol several limes; finally it was dissolved in water, and con-
centrated by evaporation on the water-bath, when a deposit
occurred. This was isolated, dried by pressure between folds of
paper, and over H2SO4 in vacuo, and finally at 100° C. At 110" C.
it was not afiected ; it was now analysed.
leading to formula C3HH,BaP0^.
Another specimen of barium glycerophosphate was prepared
much in the same way, but with this difference, that whereas the
one, the analysis of which has just been given, was separated
from water by boiling, the one now to be described was precipi-
tated from aqueous solution by alcohol, with which it was also
washed. On isolation the precipitate contracted, became horny,
transparent, and finally fused to a thick liquid. It eventually
dried to a brittle mass. On analysis it gave :
C
H
Ba
P
O
Percents.
11-46
2-44
45-15
10-21
30-74
Atoms.
2-9
7-4
1-0
1-0
5-8
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES, 131
Percents.
C = 12-611
H = 2-933
Ba = 40-950
P = 9-266
0 = 34-240
leading to formula OgH^BaPOgjH^O.
These observations led to the surmise that the barium salt as
precipitated by alcohol was a true alcoholate. The truth of this
hypothesis was proved by the experiments now to be described.
The Alcoliolo-liydrated Barium Glyceroj)hosphate. — A quantity of
barium glycerophosphate, as prepared by the chemolysis of
kephalin with barita, was precipitated by alcohol, and the pre-
cipitate washed with alcohol, after which it was exposed to the
air, when it lost alcohol, became brown and somewhat brittle
round the edges, and began to fuse. At this stage it was again
dissolved in the minimum amount of cold water, and the solution
reprecipitated by absolute alcohol. The precipitate so prepared
was isolated and allowed to drain. When thoroughly drained, a
small quantity was pressed in a vice between folds of blotting-
paper, until it became pulverulent. It was now heated in an air-
bath at 100° C, until it was approximately dry. In this way
0-7354 g. lost 0-237 g. and became 0*4978 g., corresponding to a
loss of 32-30 per cent. The residue of this operation, when burnt,
left a residue weighing 0*3064 g., equal to 61-5 per cent.
Pure normal barium glycerophosphate leaves on ignition 72*96
per cent, residue, while the hypothetical acid salt of formula
C3H7BaPO^,C3HgPO,3, might presumably leave under such con-
ditions 65*3 per cent, of H2BaP20^.
A salt of the formula qI^jj'^^pq*" | -^2^ would leave on igni-
tion 62*9 per cent. H^BagPO^.
After this preliminary experiment, the whole precipitate ob-
tained by alcohol, amounting to 71 g., was dried by pressure
between folds of paper, until it was quite pulverulent. It was
then placed in 200 cc. water, in which it set like glue, but after
forty- eight hours had not entirely dissolved. An addition, however,
of 100 cc. more water produced a perfect solution. This was now
subjected to distillation, and the alcohol contained in the first
150 cc. distillate determined. It was thus shown that in the 71 g.
of glycerophosphate, which had been so dry that it could be
9—2
132 CHEMICAL CONSTITUTION OF THE BRAIN
powdered, there were 14-84 g. of absolute alcohol, or 20-9 j^er
cent.
The analytical results show :
Absolute ethylic alcohol - - - 20-9
Water - 11-4
Total volatile at 100° C.
Eesidue of barium phosphate -
Volatile at red heat
Total glycerophosphate of Ba -
- 32-3
32-3
- 41-6 (form undetermined.)
- 26-1
- 67-7
67-7
100-0
There are probably at least three molecles of alcohol and six
of water combined with one molecle of acid glycerophosphate in
this compound."^
The residue, from which the alcohol had been distilled, was
concentrated by evaporation on a water-bath, when it deposited
granular matter, which on analysis was found to be normal gly-
cerophosphate of barium, CgH^BaPO^^.
The mother-liquor obtained after the separation of the normal
barium salt just alluded to, was again precipitated by absolute
alcohoL The precipitate was dried by jjressure between folds of
paper until it was pulverulent. It now w^eighed 63 g., and was
dissolved in 300 cc. water, and the solution distilled to one-half.
On estimation of the alcohol, it was found that the salt had con-
tained 15*5 per cent, absolute alcohol.
Finally the residual solution of barium glycerophosphate was
transformed into lead salt, and the lead salt into calcium salt.
That portion of the calcium salt which was deposited from a
boiling solution was found to be normal. From the mother-
liquor, which grew acid, a salt was precipitated by alcohol, which,
from a determination of the residue left on combustion of a por-
tion of it, seemed to be the acid salt of calcium.
'•■ The salts of kryptophanic acid (from urine) and kreatylic acid (from
flesh) present characters which recall those of glycerophosphate of barium.
Thus the copper salt of kryptophanic acid, when precipitated by alcohol,
behaves like the glycerophosphate of barium already described, yielding, on
distillation with water, alcohol.
The f»jll()wing alcoliolates of inorganic salts are known : ZnCl2,2C2HgO ;.
CaCl,,4aHoO ; Mg(N()3),„6C,,H60 ; etc.
PHOSPHORISED PRINCIPLES OR PHOSPHATIDES. 133
Barium glycerophosphate in a concentrated syrupy sokition in
water, when allowed to stand in a covered deep vessel, crystallizes,
after the lapse of a very long time, in radiary masses of needles.
Glycerophosphate of barium, not only seems to be the only
organic compound which is known to form alcoholates, but it is
also in so far unique, as it forms an alcoholate and a hydrate at the
same time.
The differences observed in the relative amounts of alcohol and
water may be caused by the different proportions of these bodies
which are present at the moment of precipitation. If several
alcoholo-hydrates are producible, the method of preparation makes
it unavoidable that a mixture of these should be produced. But
even if this were not the case, and if there were only one type of
alcoholo-hydrate, the varying amounts of alcohol in different pre-
cipitations would compel us to assume that alcohol and water
may substitute each other in indefinite proportions, as isomor-
phous compounds do in mixed crystals.
The insoluble lead salt is very stable ; next comes the calcium
salt, then the barium salt.
Other salts, such as those of silver and copper, seem to decom-
pose at every stage of their production, so that although volumi-
nous at first, they fall away to almost nothing during attempts
at their purification.
During all transformations or concentrations of solutions, con-
siderable quantities of the acid are decomposed, and the relative
phosphates and glycerol are formed.
134 CHEMICAL CONSTITUTION OF THE BIIAIN.
lY.
GROUP OF NITROGENISED NONPHOSPHORISED
PRINCIPLES.
A. SUBGROUP OF THE CEREBROSIDES.
General Proioerties of the Stihgrovp.
The cerebrosides are all white, and more or less opaque, but
are capable of becoming in part transparent like wax. They are
deposited from hot alcoholic solutions in minute microscopic
particles, which may be termed crystalline, but have no claim to
be termed crystallised. These particles are arranged in various
composite forms — balls, or branched masses, or rosettes ; the
latter will be more fully described under the headings referring
to the several varieties.
The cerebrosides are all soluble in hot alcohol, particularly
absolute alcohol, and deposited on cooling ; they are very little
soluble in cold absolute alcohol, much less soluble, indeed, than
sphingomyelin, which can thus be separated from the bulk of the
cerebrins. The mixture is dissolved in hot alcohol and allowed
to cool ; nearly all the cerebrin bodies fall down ; much sphingo-
myelin remains in solution. The deposit is separated from the
liquid and subjected to this treatment until the mother-liquor is
free from phosphorus. It is further purified, as will be shown
lower down.
The cerebrosides are almost insoluble in water. One g. of purified
phrenosin from ox was powdered and boiled in 100 cc. of water ;
the solution was filtered through force-filters, and of the filtrate
50 cc. were evaporated on the water-bath to dryness in a platinum
dish. As this increased in weight by only 0-025 g., one part of
this phrenosin was soluble in 2,000 parts of water. Pure phre-
NITROGENISED NONPHOSPHOIUSED PRINCIPLES. 135
nosin does not swell on being boiled with water, but remains
pulverulent and unaffected. When the cerebrosides swell and
become starchy in hot water, they contain phosphorised matter as
an admixture.
The cerebrosides are quite insoluble in cold benzol. They swell
in it, and become quite transparent, so as almost to disappear
from sight. But when the benzol is filtered off and evaporated,
it does not leave a vestige of matter behind. But in hot benzol
the cerebrosides are extremely soluble, and on cooling are de-
posited as a gelatinous mass, which requires agitation before it
can be filtered, From the hot solution cold alcohol precipitates
white flakes. This treatment facilitates the separation of the
cerebrosides from the benzol.
The cerebrosides are almost insoluble in either cold or hot
ether, and are by this solvent purified from kephalin and its
relations, from lecithin, cholesterin, from krinosin and istarin.
But the separation from myelin and sphingomyelin cannot be
effected so easily by ether as by the absolute alcohol treatment
above described, the lead-jDrocess to be described, and the cadmic
chloride and hydrothion in ethereal solution treatment to be
described lower down.
The cerebrosides behave neutrally towards hydrothion, whether
they are suspended in alcohol or ether. Any metals which may
be combined or mixed with them may then be removed as sul-
phides without injury to the cerebrosides. They are, therefore,
in this respect unlike some of the phosphorised principles, which
seem to combine with hydrothion, and are with its aid able to
retain metallic sulphides in solution in ether.
The mixture of cerebrosides has been repeatedly examined as
to the relative amount of the elements of which it is constituted,
and there have been found in percents. :
0
65-7
66-1
67-01
65-41
66-35
H
10-8
11-0
11-06
10-96
11-01
N
2*2
3-19
3-00
2-43
OP
19-1
20-7
18-74
20-63
2021
A certain amount of sulphur is also present as a constituent of
a subgroup, which will be described lower down.
The nitrogen is rarely found exceeding 3 per cent. ; so much
as 4-4 per cent, has been found only once in my researches, and
that in a case where the barita process had been used. In pro-
irs CHEMICAL CONSTITUTION OF THE BRAIN
cesses in which barita was not used, the cerebroside mixture con-
tained from 2-4 to 3-2 per cent, of nitrogen. And in a process
Avhere barita had been used, but probably in a lesser quantity, or
with less effect, only 2-2 per cent, of nitrogen was retained in
the cerebroside mixture.
]. Separation of the Cerebroside Principles of the Brain.
Spirit Treatment. — The white matter obtained by the process
described above, from which lecithin and kephalin have been
removed by treatment with ether, and which has been recrystal-
lised from spirit eight several times, is further purified as follows :
The mixture, containing phrenosin, kerasin, cerebrinic acid,
and sphingomyelin, is brought to the consistence of cream by
rubbing in a mortar with spirit of 85 per cent., and is added in
small quantities at a time to hot spirit in a platinum vessel.
This is done in order to effect as perfect a solution of the soluble
part, and to prevent as much as possible the formation of in-
soluble matter. The solution is filtered hot and set aside to
deposit. On cooling a white body comes down and is collected.
On analysis this is found to contain about 0'856 per cent, of phos-
phorus. This process is repeated a tenth time, and the product
having lost only little of the phosphorised ingredient (which in
spirit of 85 per cent, strength has the same solubility as the cere-
brin bodies) is subjected to treatment with lead acetate.
Lead Acetate Treatment. — The body is triturated with alcoholic
solution of lead acetate, and the mixture poured as before into
hot spirit ; the solution is filtered hot, and allowed to cool. The
deposit is collected, and treated with more alcoholic solution of
lead acetate, and filtered from the excess of liquid. After this it
is twice dissolved in hot spirit to remove excess of lead acetate.
The deposit, on analysis, is found to contain about 073 per cent,
of phosphorus.
The body is now triturated with icatery solution of lead acetate,
and pressed to remove the excess of liquid. The mass is made
into a cream with cold spirit, and added in small quantities to hot
spirit. The solution is filtered and set aside to cool, and the
stearoconotiscd portion is put aside for separate treatment. The
alcoholic and watery filtrates, from which all matters deposited
on cooling have been removed, are concentrated, and treated as
shall be described elsewhere. The white body which comes down
NITROGENISED NONPHGSPHORISED PRINCIPLES. 137
from the spirit solution on cooling is collected and treated again
with spirit, and this process is repeated until no more so-called
stearoconote (or lead ^precipitate) is produced. The ultimate
deposit obtained, after all these recrystallisations have been
carried out, is collected and dried at 100° C. At this temperature
it does not alter in appearance. It gives the purple reaction with
oil of vitriol alone, and on analysis is found to contain 0*73 per
■cent, of phosphorus.
Lead acetate, without ammonia, as here applied, precipitates
almost all true myelin, besides some cerebrinic acid and other
matters. To purify the cerebrosicles completely from matters of
this class, it is necessary to add ammonia to the lead acetate.
By that means a condition of the alcoholic solution of the cerc-
brosides is attained, in which neither lead acetate, nor ammonia,
nor a mixture of both produces any further precipitates. Then
the solution contains mainly phrenosin, kerasiu, krinosin, and
sphingomyelin, which are deposited on cooling, excepting only
some kerasin and sphingomyelin, which .remain in solution.
Absolute Alcohol Treatment ivitliout Fractionation of Precipitate. —
Absolute alcohol is now used in place of spirit of 85 per cent.,
and the solution and recrystallisation are repeated a great number
of times until the rnother-liquors gives no more precipitate with
cadmic chloride. As long as the alcoholic filtrate gives a precipi-
tate with this reagent it is manifest that it removes the phos-
phorised sphingomyelin, and, as will be seen below, the separation
of the phosphorised part succeeds, gradually but effectually, to
the extent of concentrating the phosphorus in the part soluble in
the cold, so that it contains nearly 2 per cent. (1-951 per cent.),
while the part insoluble in the cold retains only one-tenth of that
amount. But when the solubility of the phosphorised ingredient
in absolute alcohol has become again equal to that of the nitro-
genised substance, it is found requisite to resort to fractional
precipitation for the purpose of isolating pure educts.
Separation of Phrenosin and Kerasin by Fractional Precipitation on
Cooling. — When the absolute alcohol solution begins to deposit
matter, which occurs between 50' and 40°, rosettes of phrenosin
appear first. When the temperature reaches 28° this ceases ; and
the supernatant liquor is clear for a while until the temperature
falls to 26°. Below this tempei'ature a gelatinous cloudy mass,
mainly of kerasin, gradually forms and floats on the phrenosin.
138 CHEMICAL CONSTITUTION OF THE BRAIN
The phrenosin is therefore isolated as follows. When the
temperature of the liquid in which the phrenosin crystallises has
fallen to 28°, the mother-liquor which contains most of the kerasin
in solution is swiftly decanted, either with or without the employ-
ment of a filter kept at 28° by a water-bath ; and the phrenosin
is thus recrystallised seven times until no further separation seems
to be effected.
An analysis of a large specimen of phrenosin at this stage gave
phosphorus equal to 0-182 per cent., and after two further treat-
ments in the same way it yielded phosphorus equal to 0*1 13 per
cent. It also gave in two estimations the following quantities of
inorganic matter : (I.) =0=19 per cent. ; (II.) = 0-23 per cent., con-
taining "07 of potash (KgO). The dried kerasin yielded phos-
phorus = 0*198 2)er cent, and potash =0-07, from which it will be
seen that the many resolutions and the several lead-treatments
had not yet removed all inorganic ingredients from these matters.
2. Phrenosin and its Derivates.
a. Further Purification of Phrenosin hij Cadmic Chloride, Ether, and
Hydroihion.
The phrenosin isolated by fractional precipitation as described
above is mixed with solution of cadmic chloride, and the
mixture suspended in ether. Hydrothion is j)assed into this,
when a yellow solution and a yellow precipitate form, which
remain mixed with the bulk of the undissolved phrenosin. The
solid matter is filtered from the liquid ; it consists of phrenosin
and cadmic sulphide, while a peculiar compound of a phosphorised
body with cadmic sulphide remains in solution. The residue is
removed from the. filter and dissolved in hot 85 per cent, spirit.
The insoluble cadmic sulphide is filtered off, and the solution is
set aside to deposit. On cooling phrenosin comes down in large
rosettes, and is collected and recrystallised, first twice from 85
jDer cent, spirit, and finally from absolute alcohol. It is dried^
and then exhibits the following properties. When boiled with
water it does not swell to a starchy paste, but merely becomes
flocculent and floats about in the fluid. Treated by itself in the
cold with oil of vitriol it very slowly develops a purple reaction,,
but more quickly when warmed. It passes through an interme-
diate yellow stage, particularly well marked in the reaction, which
is obtained without the employment of heat, and during which
NITROGENISED NONPHOSPHOPJSED PRINCIPLES. 13i>
the matter is wholly in solution. Then flocks separate and slowly
become purple.
Two quantations of phosphorus were made : No. 1 gave 0-045
per cent., and No. 2 gave 0*051 per cent. Taking the mean of
these two results, we have 0 048 per cent, of phosphorus. Cal-
culated as myelin, this would give 1 per cent, as the amount of
phosphorised substance left in the phrenosin.
At this stage of its purification the phrenosin was subjected to
elementary analysis.
Elementary Analysis of Phrenosin, C^^^Hw^NOg. — Carbon, hydrogen
and nitrogen were determined simultaneously by the vacuum
method. The substance was burnt with oxide of copper and copper
in vacuo, the resulting water was weighed, and the gaseous mixture
of carbonic acid and nitrogen was analysed and estimated volu-
metrically.
The nitrogen was further determined by the methods of Liebig
and of Dumas as modified by Thudichum and Wanklyn, and
by the proceeding of Will and Varrentrapp. The percentages
obtained in these analyses are compared in the following table :
By combustion in
By combustion in vacuo ; C CO2 atmosphere; By combustion with
and N volumetrically ; CO2 gas volumetri- soda-lime ; Pt salt.
HoO weighed, wgd. cally estimated. weighed.
a. h. c. d. e. f. g. h. i. k.
C 67-71 67-89 67-37 68-56
H 11-62 11-42 11-23
N 2-15 2-13 2-07 2-29 2-18 2-34 1-768 1-690 1-715
0 18-51 18-58 19-03
100-00 100-00 100-00
Consideration of the Methods of Analysis. — The quantation of the
nitrogen as gas, whether it has been obtained by combustion in a
vacuum or by combustion under atmospheric pressure in an
atmosphere of carbonic anhydride, always gives the nitrogen a.
little too high as compared to theory, whereas the estimation of
nitrogen by transformation into ammonia always gives the amount
of this element a little lower than is required by theory. These
discrepancies are well known to be inherent in the methods.
They are less significant as regards the analysis of bodies which
are rich in nitrogen, than in the analysis of bodies containing only
a small percentage of this element. In the particular case of
140 CHEMICAL CONSTITUTION OF THE BRAIN.
phrenosin they are so great as to make it at first sight impossible
to derive a correct empirical formula from the data given by either
mode of analysis. But the mean of the data for nitrogen obtained
by the three different modes of analysis is very nearly coincident
with the sum of the data obtained by the chemolytic method.
This coincidence is probably the result of accident only, at least
there is at present no explanation derivable from the most search-
ing scrutiny of either of the methods employed, or of the parti-
cular manner in which they ha^^e been executed.
In the vacuum analysis the carbon is regularly found a little
too loAv j yet not a trace of carbon is left unburned in the tubes,
as was specially proved ; moreover, the combustion tubes with
their contents were, weighed, before and after combustion, and
"the weights of the sums of the ])roducts of combustion, as calcu-
lated from their volumes in the case of carbon and nitrogen,
showed an almost mathematical coincidence with the respective
losses which the tubes had undergone.
In this case of phrenosin the known methods of elementary
analysis are therefore unavailing to lead to final results ; on the
contrary, phrenosin is a good test object by the use of which the
particular failings as well as strong features of these methods can
be made apparent. Thus, combustion, according to Liebig, of
not too small a quantity of phrenosin j'ields the best carbon esti-
mate ; combustion in vacuo of such a small quantity as that to
which the method is necessarily limited, the worst.
The very same methods which in the case of phrenosin yield
the results discussed in the foregoing, give, when applied to its
decomposition-products, results which are in much greater con-
cordance with the requirements of theory. This unquestionable
fact shows that the size of the molecle, and the proportions in
which the elements contained stand to each other, have an influ-
ence on the result of the process of elemcntar\^ analysis ; a simple
compound of fewer atoms gives more accurate results when tested
by different methods, while a more complicated compound with
many atoms, when tested in the same manner by different methods,
gives much less accurate results. It is in accordance with this
that the elementary analyses of the more com})licated nitrogenised
bodies, such as kerasin and cerebrinic acid, present even greater
difficulties than those which have been experienced in the analysis
of phrenosin.
NITROGENISED NONPHOSPHORISED PRINCIPLES. 141
1). Chemolysis of Phrenosin hy Sulj^huric Acid in Watery Solution.
Introduction. — In my earliest chemolyses with barita, hermeti-
cally sealed glass tubes, enclosed in iron tubes, were employed.
But of these at least half succumbed to the internal pressure, and
their contents were lost. I therefore procured a special tube of
brass lined with platinum. This worked satisfactorily for the
barita chemolyses, which were effected in a short time ; but the
sulphuric acid chemolyses were found for their completion to
require the influence of a temperature of at least 130° for a period
varying from 310 to 370 hours. It was therefore necessary to
multiply the tubes, and in order to do this consistently with
practical considerations leaden tubes were employed. Such tubes
could not have been employed in the barita chemolysis, as barita.
rapidly and energetically attacks lead. But dilute sulphuric acid
has but a slight influence on metallic lead, and the small quantity
of metal which is dissolved is easily removed from the organic
products by appropriate means.
The Apparatus. — The Leaden Tubes. — The Hot-Air Stove. — The
leaden tubes are an inch in calibre ; the metal is an eighth of an
inch thick ; each tube is eighteen inches long. One end of the
tube is closed by hammering only so as to form a semi-globular
end, not larger in diameter than the tube itself ; it is tested by
water, and when none passes it is soldered over on the outside.
The mixture to be chemolysed is now put into the tube by means
of a wide-tubed funnel ; the upper part of the tube is heated to
dry it completely and rarefy the air. in the air-space, which
amounts to about one-sixth of the length of the tube ; the mouth
of the tube is now suddenly compressed in a vice and closed ; the
edges of the lead are filed smooth, moistened with zinc chloride^
and immediately soldered, or, as the operation is technically
termed, ' burnt,' with the oxyhydrogen flame.
The hot-air stove is made of copper, and consists of tivo horizon-
tal air cushions, between which special room for six of the leaden
tubes just described is arranged so that their longest axes are
lying in a horizontal position. The tubes can thus be kept at an
equable temperature, which can be read by the thermometer, the
ball of which is in the central air-space, while the stem projects
over the top of the stove. The stove is heated by aerated gas-
flames.
142 CHEMICAL CONSTITUTION OF THE BRAIN.
FreHminarij Piuificatioa from Inorganic Salts of the Phrenosin to
he Chemolijsed. — As phrenosin retains inorganic salts with great
pertinacity, it must be subjected to a process for the removal of
these. It is boiled in water until completely disintegrated, and
the boiled mixture is pressed through a cloth. The filtrate is
then mixed with sulphuric acid sufficient for it to contain 1 per
cent, and boiled for one hour. The phrenosin is soon curdled out
of the solution, free from salt but partially altered. For the acid
solution after treatment with barita carbonate is found to contain
some chemolytic sugar (cerebrose) besides some alkaloidal matters,
and the potassium, sodium, and earthy salts which it is the object
of the process to remove.
Chemolysis of the Purified Substance. — Six leaden tubes, prepared
■as above, receive each about 30 g. of nitrogenised substance
purified as described, and 353 cc. of dilute sulphuric acid contain-
ing 2 per cent. H^SO^. The tubes are then closed as described,
placed in the hot-air stove, and heated to 130^ during twenty-
four hours. After the lapse of this period the tubes are opened
at the compressed end with "a chisel, the dilute acid is removed
and filtered, all solid matter is kept in the tubes cr returned to
them together with a fresh charge of acid ; the tubes are again
closed as described, and heated for a second twenty-four hours.
This treatment is repeated as long as the acid liquid contains
any cerebrose, and the chemolyses are deemed complete only
when the last charge of dilute acid is found on proper treat-
ment and concentration to be free from cerebrose. This result
is in most cases not attained in less than fourteen days, and
in some requires from sixteen to seventeen days, in a few even
twenty-four, during which the chemolysis is continued day and
night.
The Acid Filtrates. — These are boiled w^ith barium carbonate,
prepared pure by precipitation for the purpose. The neutral
filtrates are evaporated to about one-fifth of their bulk in a water-
bath, then removed into a distilling apparatus connected with an
air-pump, and distilled at a temperature of from 30' to 40° to the
consistency of a syrup. The latter is put aside to crystallise.
Stellate groups of crj'stals soon form, and the entire syrup
gradually solidifies to a granular mass of crystals. These are
separated partly by drawing off the small quantity of mother-
liquor, partly by agitating them with water, and getting rid of
NITROGENISED NONPHOSPHOPJSED PRINCIPLES. 143
the mother-liquor by dilution ; for the crystals are but slowly
soluble in cold water.
From the mother-liquor a further quantity of crystals is ob-
tained by addition of boiling alcohol to the hot concentrated
aqueous solution until a considerable permanent turbidity is pro-
duced. When the mixture is alloAved to stand in the cold for
one day, it forms a considerable amount of a coloured deposit,
from which the clear supernatant alcoholic liquid is decanted.
This latter solution on standing for some weeks, and after re-
j^eated additions of small quantities of alcohol, and lastly of ether,
deposits a crop of white crystals which are added to those
obtained in the first operation.
The mother-liquors are, however, always considerable in amount,
and after concentration yield an uncrystallisable syrup, which
amounts to about the same weight as the crystals obtained from
it. The syrup can be made to crystallise a third time after the
removal of some traces of potassium and of a compound am-
monium base by platinic chloride, and the removal of all traces
of the reagent by hydrothion and silver carbonate.
c. Cerebrose, a New Crystallised Sugar.
The Crystals. — Cerebrose CgH^^O^. — The crystals obtained as
described in the foregoing are dissolved in water, and the solution
is evaporated in vacuo, after treatment with animal charcoal. A
mass of crystals is again obtained, which are smaller than the
first ones, but perfectly white and very hard. The colourless
mother-liquor of these on standing over .oil of vitriol solidifies to
.a hard mass of crystals, which rise much over the level of the
liquid in which the}^ form. The crystals are not large enough
for crystallometric treatment ; seen under the microscope they
seem to consist of rhombic octahedra, of which some are elongated
to prisms. It is a kind of sugar, to which I have given the name
•of Cerebrose. On elementary analysis it gives analytical data,
leading to formula C^H^gOc-
Eeditcing Power of Cerebrose over Cupro-Potassic Tartrate. — Cere-
brose reduces Fehling's solution readily on heating, and the
precipitated suboxide of copper is mostly of a dark-red colour.
■0-2622 g. cerebrose dried in the water-bath were dissolved in
water, and the solution made to fill the space of 50 cc. This
solution therefore contained 0-5245 per cent, of cerebrose. It
144 CHEMICAL CONSTITUTION OF THE BE A IN
was employed to reduce a Fehling's solution of which 5 cc. re-
quired 0*025 g. of glucose for decoloration. Five cc. of the
Fehling's solution required 5-6 cc. of the cerebrose solution for
decoloration, equal to 0-0294 g. cerebrose. A certain quantity of
cupro-potassic tartrate, therefore, which requires five parts of
glucose for complete reduction, requires about six parts of cere-
brose for reduction.
Polarising Poicer of Sohttion of Cerebrose. — Some cerebrose was-
dried in the air-bath at 90°, and in vacuo till it remained constant
in weight. Of this 2-6315 g. were dissolved in water with the
aid of a gentle heat, and made to fill a space of 20 cc. The solu-
tion was treated with animal charcoal to remove a slight turbidity.
Its strength remained at 13-16 per cent. This in a tube of 100 mm.
length rotated the ray of polarised light at 25° T= +10° 40' ; but
after twenty-four hours' standing the same tube at T 11° rotated
only + 9° 24'; after a second twenty-four hours, at T 12°= -|- 9° 18'.
The solution was then diluted with an equal bulk of water, and
its rotation, measured in a tube twice the length of the former^
was found to be + 9° 24' at 9° T to 9° 32' at 8° T. These data,
by means of the usual calculation, lead to the specific or limited
rotation for cerebrose of + 70° 40'. It will be observed that im-
mediately after solution the rotation is a little higher than
twenty-four hours after solution, when it becomes constant. But
this increase of the rotation is very slight compared with the in-
crease which dextroglucose exhibits immediately after solution,
and which, from the fact of its being about as much again as the
constant rotation, is termed the birotation of glucose.
The molecular formula of cerebrose is assumed in the foregoing
to be C^3HjoO,3. Cerebrose resembles sugar of milk by its feebly
sweet taste and the great hardness of its crystals.
Other Properties if Cerebrose. — The crystallised cerebrose is never
obtained without the amorphous modification being formed at the
same time. To judge from comparison of bulks, at least half the
cerebrose obtained during the chemolytic operations on the nitro-
genised principles passes into the amor2)hous state, and cannot be
made to crystallise entirely even within the period of a year.
Crystalline particles of pure cerebrose immersed in the syrup in-
crease in size, and form ramifications ; but their growth ceases
after a time,, and the syrup thereafter remains unchanged. The
difficulty of separating the two modifications is increased by the
NITROGENISED NONPHOSPHORISED PRINCIPLES. 145
occasional appearance of a third form of product of the chemolytic
metamorphoses of the amylonide radicle of the nitrogenised bodies
which I shall presently describe.
Cerebrose is precipitated from its watery solution by basic lead
acetate or by a mixture of neutral lead acetate and ammonia ; the
mother-liquor of this precipitate no longer reduces potassio-cupric
tartrate, from which it may be inferred that the precipitation of
the cerebrose is complete. The lead compound of cerebrose after
decomposition by hydrothion yields the cerebrose in the free state.
In its affinity for lead oxide cerebrose resembles inosite, the sugar
naturally contained in the brain, and obtained as an educt from
the water extracts ; but it is easily distinguished from inosite by
its power over polarised light and potassio-cupric tartrate, reactions
which inosite does not possess.
The Uncrystallised Cerebrose. — The uncrystallised cerebrose ob-
tained from the nitrogenised substances by the process of
chemolysis above described may be a product from the crj^^stal-
lised j at least, when the watery solution of the sugar is evapo-
rated in the open air on the water-bath no crystallised sugar is
ever obtained, but, as previous experience and renewed experi-
ment have shown, only uncrystallisable cerebrose. Only w^hen
the solution is evaporated in a vacuum ensuring the absence of
air, and at a temperature never rising above that of the animal
body, 37°, is crystallisable cerebrose obtained — accompanied,
however, always by a considerable proportion of uncrystallisable
cerebrose, amounting in weight to about that of the crystallisable
cerebrose. There is, therefore, room for an inquiry into the
causes of these phenomena.
The chemical constitution of cerebrose now arises as a subject
of inquiry of interest and importance. Not only is there a new
isomer added to the long list of saccharoid substances already
known, but, what is of much greater value, a new key is found to
the knowledge of the constitution of some of the organoplastic
substances. This will enable us to obtain a full knowledge of the
constitution of the nitrogenised substances of the brain much
quicker than would be the case without such theoretical aid ; for
the number and nature of the problems are now at once limited
and defined, as we shall see presently by the aid of further new
data.
Cerebrosic Acid, C^fl-^Q('H.2)0^^. — This new acid, obtained by
10
146 CHEMICAL CONSTITUTION OF THE BRAIN.
means of the chemolytic process from phrenosin, has the compo-
sition of a carbohydrate, and is probably isomeric with cerebrose.
It has not -yet been examined any closer in the free state, but the
examination of its barium salt leads to the inference that it is a
dibasic acid of the formula C,3Hj.20j3. It is obtained as folloAvs :
25 g. of pure phrenosin are suspended in 300 cc. of water, and to
the mixture 2 cc. of oil of vitriol are added. The whole is en-
closed in the platinum chemolyser, and heated to 120° during
seven days without interruption. The acid liquid from the
chemolyser is now filtered and treated with barium carbonate at
the boiling heat. The filtrate (which in the chemolyses of
phrenosin, where the dilute sulphuric acid is renewed every
twenty-four hours, mainly contains cerebrose) has no reducing
effect at all upon potassio-cupric tartrate, but contains a con-
siderable amount of barium in solution. When evaporated quickly
to dryness, it leaves as residue a hard amorphous barium salt ;
but when dissolved in a little spirit and allowed to stand, it
slowly sets into a mass of indistinct crystals. These are freed
from mother-liquor by pressure between folds of bibulous paper
dried at 100° C, and analysed with the following result :
Synopsis of Analyses of Barium Cerebrosate.
Elements.
Percentages.
-^At wts.
c
24-53
2-0442
6-40
H
3-20
3-20
10-09
Ba
43-50
0-3175
1-00
0
28-77
1-798
5-66
These data lead to a formula BaCgHj^O^j, corresponding to an
acid of the composition of cerebrose in which two atoms of
hydrogen are replaced by an atom of barium. It reminds of
glucic acid, which is also dibasic. Glucic acid is prone to form
bodies like the caramels, and the presence of a small proportion
of one or other of these bodies seems to be the cause of an excess
of carbon over the theoretical amount which is met with in the
analysis of its salts. A similar feature is observed upon the
cerebrosate of barium, which also exhibits a slight excess of
carbon and a deficiency of hydrogen and oxygen. The salt
is free from nitrogen, and does not contain sulphuric acid in
organic combination. When distilled with phosphoric anhydride
it does not give out the odour of acrolein, but a smell resembling
NITROGENISED NONPHOSPHORISED PRINCIPLES. 147
burnt sugar. With oil of vitriol alone it gives no purple reaction.
Mixed with sphingosin and oil of vitriol it gives a brilliant purple
reaction, equal in tint to that produced by the aid of cerebrose.
But even its concentrated solution has no influence on the alkaline
copper solution.
Transformation of Baiium Salt of Cerebrosic Acid into Zinc Salt. —
The barium salt is decomposed with dilute sulphuric acid, and the
solution extracted with ether. The ether, on distillation, leaves
an acid which is converted into zinc salt : the latter is crystallised
and analysed. The air-dried salt loses 9-8 per cent, of water at
100° C. The remaining dry salt is burnt, and the zinc estimated,
from the remaining oxide, to amount to 28 per cent, of air-dried
salt. From these data it seems that the zinc salt is entirely
different from any of the known lactates. The barium salt,
though different in appearance from lactate, is isomeric with it.
Attempt to produce Cerebrosic Acid from Free Cerebrose. — A con-
sideration of the conditions under which this acid has been pro-
duced from phrenosin suggests that it might have been formed by
the prolonged action of heat and acid upon cerebrose formed
during the earlier stages of the reaction. In the experiments
in which cerebrose was obtained, the influence of acid and heat
upon the phrenosin was interrupted every twenty-four hours, and
the acid was renewed. In the experiment, however, which yielded
cerebrosic acid, the action of acid and heat had been continuous
during seven days and nights. It was therefore necessary to in-
vestigate whether cerebrose already formed could be transformed
into cerebrosic acid under the circumstances related, or whether it
could be so transformed only in the nascent state, and what were
the other conditions of this transformation.
About 2 g. of amorphous cerebrose were heated with water
containing 1 per cent, of oil of vitriol, to 120° during nine days.
There were formed, firstly, a considerable amount of caramel ;
and secondly, a quantity of an acid corresponding, as regards its
properties and those of its salts, to cerebrosic acid. But a large
proportion of the cerebrose remained unchanged. The caramel
was, like the acid, soluble in ether.
Caramel obtained in the Chemolysis of Phrenosin in which Cere-
brosic Acid was formed. — Of this caramel 2-1 g. were obtained. It
was very soluble in ether, and somewhat soluble in alkaline water,
but insoluble in alcohol and in acidulated water. It was of a deep
10—2
148 CHEMICAL CONSTITUTION OF THE BBAIN
brown colour, like all the bodies of this class. From a considera-
tion of the several forms of caramel which are obtained from the
several principles of the nitrogenised group, as will be shown in
another chapter, it becomes probable that the caramel here formed
was, in part at least, the caramel of psychosin. This supposi-
tion is strengthened by the consideration of the relative quantities
of the several products of the chemolysis of phrenosin. The 25 g.
of this body employed in the experiment related above gave 4-6 g.
of cerebrosic acid. The theoretical amount which could have
been obtained in the best case would have been 6*2 g. Con-
sequently 1 -6 g. of the amylonide radicle must have been trans-
formed into bodies other than cerebrose and cerebrosic acid. The
2-1 g. of caramel found in the present experiment could not
have been caramel of cerebrose simply, as the atomic weight of
that body is much smaller than that of cerebrose. But it could
have been, or contained, some caramel of psychosin, the atomic
weight of which is more than twice that of cerebrose. The other
products insoluble in water were, sphingosin, which as sulphate
weighed 2-5 g., corresponding to about 2*13 g. free sphingosin,
and an unascertained quantity of another alkaloid, probably
psychosin, which remained in the alcohol from which the sphin-
gosin was precipitated by sulphuric acid. The amount of free
fatty acid obtained was 9*4 g. This was almost entirely
neurostearic acid, and its quantity came very near to that
required by theory, which is 9-9 g. The total of weighed
products of chemolysis amounted to 18*2 g. ; the psychosin or
second base was not weighed, and allowing for this, certainly a
few grammes, and much for loss in the many difficult manipula-
tions, the fate of the phrenosin originally employed is pretty well
accounted for.
In almost all chemolyses of nitrogenised principles by acids or
alkalies in watery or sj)irituous solution there has been formed
cerebrose, cerebrosic acid, psychosin, caramel of psychosin, sphin-
gosin, and neurostearic acid or its ether. When the cerebrose
solution was freed from sulphuric acid, after chemolysis with an
acid, it always retained some baryta, which had to be removed
by precipitation with sulphuric acid. It is therefore possible
that the cerebrose is always accompanied by some cerebrosic
acid ; attempts should be made to extract this by ether before
evaporating the cerebrose solution to a small bulk, as otherwise
NITROGENISED NONPHOSPHORISED PRINCIPLES. 149
the acid may contribute to transform the cerebrose into the un-
crystallisable modification.
cl. Sphingosin, a neiv Alkaloid, as Sulphate, and Fatty Acids.
The solid products from the chemolytic tubes are united, edul-
corated with water, dissolved in hot spirit, decolorised with animal
charcoal, crystallised and dried. In a state of fine powder they are
extracted with pure ether in the cold. The fatty acids dissolve,
while a body remains insoluble, which is of an alkaloidal nature,
and to which, in commemoration of the many enigmas which it
presented to the inquirer, I have given the name of Sphingosin.
The part insoluble in ether is again treated with alcohol, but
the substance, previously freely soluble in spirit, now becomes
more and more insoluble, and at last fuses and becomes quite in-
soluble even in absolute alcohol. It is now easily soluble in
benzol in the cold. Addition of any acid to the hot alcohol restores
the solubility of the body, and on cooling the body crystallises
again. This bearing leaves little doubt that the body is a salt-
like combination of an organic base with the acid employed,
soluble in alcohol in the presence of an excess of acid, insoluble
in the absence of such excess.
Removal of the Sidplmric Acid by Caustic Allccdi, — The salt is
freed from spirit by water, and then while diffused in water is
treated with caustic soda ley and heated. The flaky body at once
transforms into oily drops, which rise to the surface of the liquid.
On cooling this oily liquid does not set like a fat, but becomes
again opaque, and distributed in flakes through the fluid. The
oil is, however, easily soluble in pure ether (in which the salt had
been previously insoluble), and is extracted by this solvent. The
solution is filtered, the ether distilled off", the residue dissolved in
absolute alcohol and decolorised by animal charcoal. Minute
quantities of impurities, probably of soda-soap, which deposit
from the ether solution and from this last alcoholic solution on
standing, are removed by filtration. This alcoholic solution of
the free base, which is alkaline to test paper, gives the following
reactions. Oil of vitriol in absolute alcohol gives an immediate
white precipitate of a sulphate. Hydrochloric acid gives a pre-
cipitate of a hydrochlorate ; both precipitates are soluble in cold
alcohol with the aid of excess of acid. An alcoholic solution of
cadmic chloride gives a precipitate soluble in excess of absolute
150 CHEMICAL CONSTITUTION OF THE BRAIN
alcohol. Mercuric chloride gives a flaky precipitate which settles
easily. Water causes a gelatinous precipitate in the alcoholic
solution. Ether or alcohol solution leave the base in a crystalline
state on evaporation. It is very slightly if at all soluble in
water, even on boiling. When dry it gives no purple colour with
oil of vitriol alone on gentle warming, but on addition of sugar
gives immediately a deep purple colour. The full significance of
this reaction will be discussed lower down.
Sphingosin Sulphafe.-^A solution of the free base in cold absolute
alcohol is precipitated with a freshly made solution of oil of vitriol
in absolute alcohol with the precaution of keeping the alkaloid in
excess. The white crystalline precipitate is washed with absolute
alcohol, pressed, and dried in vacuo over sulphuric acid.
Elementary analysis leads to the formula for sphingosin sul-
phate of 2(CjK.H35N02) + H2SO^, of which the theory is compared
with the experimental data in the following table :
Theory of
f ^
Atoms. Found.
^ , Percents. Percents.
34 C 408 60-11 60-85
72 H 72 10-78 10-70
2 N 28 4-19 4-14
4 0 64 — —
668
Sjjhingosin Hydrochlomte. — On adding to a concentrated solution
of sphingosin in absolute alcohol or in spirit some hydrochloric
acid, a turbidity or precipitate is at once produced. If the mixture
is warmed and allowed to cool gradually under a dryer over oil
of vitriol, masses of spear-shaped crystals form in the fluid which
can be removed as a felted mass. The crystals under the micro-
scope appear as uniform needles with pointed pyramidal ends.
The analytical data concerning this salt show that its formula is
C17H05NO2 + HCI. It does not easily form double salts with
metallic chlorides.
The purification of sphingosin is based upon its precipitation
from absolute alcohol by sulphuric acid and the decomposition of
the sulphate in water by caustic alkali ; the oily mass when freed
NITROGENISED NONPHOSPHORISED PRINCIPLES. 151
from all alkaline liquid can be boiled with water, and thus freed
entirely from alkali. In this way two impurities which may
accompany sphingosin in small quantities are entirely removed ;
one a body soluble in ether, and which will be described here-
after ; another an acid of which also a small quantity escapes the
first extraction by ether, in which the bulk of the acid formed in
the chemolysis is separated. Both remain in the absolute alcohol
from which the sphingosin is precipitated by sulphuric acid. But
they are less soluble in watery alcohol than the sphingosin, and
therefore are precipitated by water or watery reagents together
with the sphingosin.
Consideration of the General Chemical Function of Sphingosin. — The
consideration of the molecular formula of sphingosin might at
first sight lead to the hypothesis that it contained a fatty acid
radicle of the Qj^on^^ series, and that in this an atom of hydrogen
was replaced by the amide group NH^, as expressed by the formula
Ci^H33(NH2)02. Sphingosin also behaves like an amido-acid in
this, that on the one hand it combines with bases such as potash,
or on the other unites with acids such as sulphuric and hydro-
chloric. However, its qualities as an acid are the least apparent,
and so limited that they subsist in any degree only in the absence
of water. In the presence of water the potash compound is not
formed at all ; the barium compound is formed in the presence
of water and excess of- barium salt, but decomposes during every
treatment for its purification, even by resolution in strong spirit.
The salts of sphingosin with acids, however^ are very firm com-
pounds ; they crystallise, do not dissociate in solvents, and in the
dry state admit of convenient manipulation. By its greater
affinity for acids, sphingosin indeed differs from the amido-acids
and resembles more the alkaloids. It is precipitated by most
of the specific reagents which combine with alkaloids, and there-
fore on the whole evidence it must be admitted that sphingosin
is an alkaloid.
Neutral, Acid, and Basic Salts ; Bearing of the Sidphate. — When
sphingosin sulphate is treated with dry neutral ether, it remains
undissolved. When the mixture is acidified with sulphuric acid,
the sulphate dissolves completely. When to the solution some
alkali is cautiously added, the neutral sulphate is again precipi-
tated in flocks. AVhen neutral sphingosin sulphate which has
been dried completely is digested in the cold with aqueous
152 CHEMICAL CONSTITUTION OF THE BBAIN
ammonia, sulphuric acid is dissolved, and appears in the filtrate.
But it is not ^practicable to remove all the sulphuric acid from the
sphingosin by this treatment; even after many days' washing
the alkaloid retains some sulphuric acid. This can only be re-
moved by warming the compound with caustic ley, and extracting
the free alkaloid with ether.
It follows from the foregoing that sphingosin forms neutral and
acid, and perhaps basic, salts.
Bearing of the Hydrochlorate. — The hydrochlorate of sphingosin
cr3^stallises from water or alcohol in long spear-shaped crystals.
It is much more soluble in hot water than in cold ; it is deposited
from a solution in water which also contains psychosin almost
entirel}^ if the solution is cooled to and filtered at the temperature
of melting ice. A little psychosin, however, easily remains with
the sjDhingosin, so that in analyses of the latter the carbon is
sometimes found a fraction of a per cent, too high. The admix-
ture is recognised by the purple test with sulphuric acid, and is
removed by recrystallisation of the hydrochlorate ; or in case of
the sulphate, by transformation into the free base, and repre-
cipitation from absolute alcohol by sulphuric acid. The psychosin
salts of both acids are the more soluble ones.
Sphingosin uith Fotash. — When the solution of free sphingosin,
prepared from the pure sulphate in ether, is dried by being
allowed to stand over solid caustic potash, a dense but trans-
lucent deposit in the shape of flakes and crusts, covering the sides
of the vessel and the masses of potash, is gradually formed. This
is a compound of sphingosin with potash, which is little soluble
in the anhydrous ether. It has not yet been separated from the
excess of potash. The ether retains a certain portion of this
compound in solution, and leaves it as a hard, dense, colourless
deposit on distillation. A preliminary analysis of this residue
gave 61"0-1: per cent, carbon, 9-96 per cent, hj^drogen, 4-52 per
cent, nitrogen, and 6'53 per cent, potassium. From this it is
evident that this residue is a mixture of a compound formed of
sphingosin and potassium or potash, with free sphingosin. The
potassium compound Cj-Hg^vNOo requires 12-1 per cent, of
potassium. The mixture therefore contains a little more than
half its weight of the potassium compound.
Sejmraiion of Psychosin from Sphingosin. — This may be effected
in alcoholic or watery solution. The mixed alkaloids are dissolved
NITROGENISED NONPEOSPHORISED PRINCIPLES. 153
in absolute alcohol, and a very dilute solution of oil of vitriol in
absolute alcohol is added so as to precipitate all sphingosin.
From the filtered alcoholic mother-liquor all alcohol is removed,
first by distillation, afterwards by evaporation with water. The
residue is warmed with some caustic potash, and the ley which
has taken up any sulphuric acid is decanted. The washed residue
is boiled with hydrochloric acid, in which it dissolves readily.
The solution is filtered hot, and concentrated to a suitable bulk.
It contains all the psychosin, and if the precipitation of sphingosin
by sulphuric acid had been incomplete, or an excess of the acid
had been added so as to redissolve some sphingosin as acid sul-
phate, this base also occurs in the hydrochlorate. The solution,
on being cooled to 0° C, deposits sphingosin hydrochlorate in
colourless crystals, which may be, filtered off in the cold, but
cannot be washed from excess of psychosin by water, as they
swell and make filtration impossible.
Fatty Acids and Matters soluble in Ether, being Products of the
Chemohjsis which yields SjMngosin. — The ether solution of the
chemolytic products obtained as described above, p. 149, is con-
centrated, and yields several deposits of fatty matters at several
stages. The first deposit after recrystallisation from spirit
resembles neurostearic acid in appearance. It begins to fuse
at 73°, but does not wholly fuse till the temperature reaches
81°. The body is therefore probably neurostearic acid, mixed
with a small quantity of acid of lower fusing-point. It is con-
verted into barium salt ; this is extracted with hot spirit, and the
fatty acid is again extracted from the barium salt by tartaric acid
and ether. The free acid now fuses at 80° and sets at 79°; it has
therefore lost some of the fatty acid melting at a lower tem-
perature.
The later deposits from ether and all the acids most soluble in
ether are converted into barium salts and exhausted with boiling
alcohol. There remains insoluble in spirit a barium salt, which
by treatment with tartaric acid and ether gives free fatty acid
which fuses below 50° C, and therefore diff'ers greatly from the
acid first deposited from the ether solution.
The alcoholic extracts of the barium salts on cooling deposit a
solid compound, and retain in solution a compound which, after
evaporation of all alcohol, is semi-solid on being heated with water,
and dissolves entirely in ether.
154 CHEMICAL CONSTITUTION OF THE BEAIN
We therefore obtain four principal bodies by these operations
on the chemolysed matters soluble in ether. Two form barium
salts insoluble in boiling spirit, and are — the first, mainly neuro-
stearic acid, CigH3Q02 ; the other, an acid of lower fusing-point,
not yet studied any further. Of the bodies soluble in hot spirit,
one is soluble in ether. The significance of all these bodies will
be made clear when we come to consider the chemical constitu-
tion of the non-phosphorised group of nitrogenised substances.
Each of these acids, which by their number no less than their
properties are remarkable, will then find a place in a natural
educt, and by another process in a systematic classification.
e. FsijcJiosin, its Properties and Metamorphoses.
This alkaloid was first obtained from the nonphosphorised
group of nitrogenised bodies by chemolysis with caustic barita.
The mere analysis of this body led to an empirical formula of
CggH^gNOg, which was uncontrolled by combinations. The free
body had, however, crystallised from alcohol. To fill up this void
I produced some combinations with hydrochloric acid and with
platinic chloride, but the products had the peculiarity of some of
the phosphorised bodies, namely of dissociating in the presence
and under the influence of small quantities of water. The hypo-
thetical salt would have contained 24*01 per cent, of platinum,
whereas the actual salt contained only 21*05 j^er cent. But the
carbon of the organic molecle decreased slightly in relation to the
nitrogen, and this led me to again analyse a further purified
specimen of psychosin, obtained by the chemolysis of phrenosin.
The jjsychosin was dissolved in hydrochloric acid and water,
and the solution made perfectly bright and colourless. Tlie
alkaloid was then precipitated by ammonia, washed perfectly,
dried in vacuo, and analysed.
Summary of Analyses of Psychosin.
Eirst Analyses (1876). Later Analyses (1878).
Percents. Percents. -r-by At. Wgts. -r-l3yN = l.
C 61-86 61-32 5-11 23-53
H 10-09 10-09 1009 46-46
N 2-88 3-04 -21716 1-00
O 25-17 25-55 1-597 7*35
Psychosin Sul])hate. — Psychosin is soluble in very dilute sulphuric
acid on boiling, but is deposited on cooling. The mother-liquor
NITROGENISED NONPHOSPHORISED PRINCIPLES. 155
of the deposit after twenty-four hours retains only a small amount
of the salt in solution, so that ammonia, which if not added in
excess precipitates psychosin from its solution in water and hydro-
chloric acid, hardly causes any precipitate. But phosphomolybdic
acid causes a more appreciable precipitate.
Psychosin Hyrochlorate. — When to a sohition of neutral psychosin
hydrochlorate in water an excess of strong hydrochloric acid is
added, a bulky gelatinous precipitate, much resembling hydrate
of alumina, is produced. The salt is so completely removed from
the solution that the latter, after filtration, gives no precipitate
with phosphomolybdic acid.
When a solution of psychosin hydrochlorate is kept on a
dialyser of parchment-paper floating on distilled water for eighteen
hours, some psychosin as well as some hydrochloric acid pass into
the water. The amount which passes is very small, and no
psychosin is deposited on the dialyser. Psychosin is therefore a
strong base, but at the same time it exercises its functions as a
colloid, such as it becomes in the presence of water and absence
of acids.
Psychosin precipitated from its hydrochloric solution by am-
monia and well washed, when allowed to remain in contact with
pure water during several days, becomes hydrated, and swells up
to a voluminous gelatinous mass. This paste retains water with
great force, and is most difficult to dry in vacuo over sulphuric
acid. Heat causes it to become brown at once ; even in the
vacuum it assumes colour.
Psychosin and Ammonia. — Psychosin dissolves readily in con-
centrated ammonia-water on boiling, and is deposited again on
cooling, and standing. The hot ammonia solution gives a preci-
pitate with barium chloride, which after isolation is soluble in
boiling alcohol and deposited on cooling. This compound loses
barium by all attempts at recrystallisation.
Chemolysis of Psychosin by Dilute Sulphitric Acid. — Psychosin is
enclosed in the platinum chemolyser with a sufficiency of sulphuric
acid of 2 per cent, strength, and heated to 130° during forty hours.
The sulphuric acid solution, separated from the insoluble part,
boiled with barium carbonate, etc., reduces Fehling's solution.
The solution also contains a small amount of an acid similar to
the cerebrosic acid described above. The cerebrose obtained
156 CHEMICAL CONSTITUTION OF THE BRAIN.
amounts to less than half the theoretical quantity, but is supple-
mented by a small portion of its derivate.
The solid products of the chemolysis are coloured brown ; when
warm they are in a state of semifusion. The mass is gently warmed
with soda solution and shaken with ether. It dissolves in the
ether without residue, imparting to it a deep brown colour. The
ether is distilled off and the residue treated with hot absolute
alcohol. This solvent leaves a small quantity of caramel of
psychosin as a brown mass, and dissolves a quantity of slightly
coloured matter. The solution is decolorised with animal charcoal,
and precipitated with a little sulphuric acid dissolved in absolute
alcohol. The precipitate is isolated, washed with ether, and dried.
It gives no purple reaction with oil of vitriol alone, thus proving
the absence of psychosin, but with a solution of cane-sugar or
cerebrose the sulphuric acid solution gives a brilliant purple
reaction. The precipitate is therefore sphingosin sulphate.
The alcoholic mother-liquor of the sphingosin sulphate contains
some matter in solution, which gives, after removal of the alcohol,
a purple reaction with oil of vitriol by itself. It is some psychosin
which has resisted the chemolytic action of the dilute sulphuric
acid.
The chemolysis of psychosin by sulphuric acid therefore takes
place according to the equation :
c^^o, + H,o = cyiiA +
Psychosin. Cerebrose. Sphingosin.
Purple lleadion with Oil of Vitriol. — I have shown that all the
cerebrosides give with oil of vitriol, on gentle warming, a reaction,
which consists in the formation of a deep purple colour. This
reaction, which, when produced with cane-sugar and oil of vitriol,
is known as Raspail's or Pettenkofer's reaction, and was for a
long time believed to be specific to biliary acids, has been shown
to be common to a large number of bodies which probably have a
radicle in common. But most of these bodies, e.g. oleic acid,
require the addition of sugar, and do not give the purple with
oil of vitriol alone. Now, the known cerebrosides are distinguished
from the biliary and fatty bodies by the faculty of giving the
purple reaction under tAvo different sets of conditions. They
give it with oil of vitriol alone, on gentle warming, after a little
time of standing ; and they give it quicker if with the oil of
NITROGENISED NONPHOSPHOPJSED PRINCIPLES. 157
vitriol a little sugar is at once added. Now we know that all
these bodies contain the radicle of the sagar cerebrose, which, as
I have shown, gives with pure gtykocholic acid and sulphuric
acid a brilliant Pettenkofer reaction, and is therefore capable of
replacing cane-sugar to its full value in this process. It is there-
fore clear that the ability of the cerebrin bodies to give the purple
with oil of vitriol alone has for one of its causes the presence in
their constitution of the radicle of cerebrose. That they react
slower with sulphuric acid alone than with sulphuric acid and
sugar added is perhaps explained by the facts evolved in the
chemolysis above described, namely, that the splitting off of cere-
brose from the other radicles requires time.
We are now able to advance the hypothesis, which has a high
degree of probability, that every phrenosin-like body which gives
a purple reaction with oil of vitriol alone contains the radicle of
cerebrose besides that other radicle which, with any sugar, cere-
brose, or cane-sugar, gives the purple, and which we will term
the oleo-cholide radicle. On the other hand, any phrenosin-like
body which does not give the purple with oil of vitriol alone may
contain either the cerebrose or the oleo-cholide radicle. If, on
addition of sugar, it gives the purple, then it contains the oleo-
cholide radicle ; if it does not give the purple, then this radicle
also is excluded.
I will now proceed to the application of these data to the test-
ing of the chemolytic products above described. Sphingosin,
with oil of vitriol at a very gentle heat, dissolves and becomes a
little yellow. But no purple colour is produced. On addition of
cane-sugar or of cerebrose in highly concentrated solution the
purple is immediately struck. Psychosin, with oil of vitriol at a
very gentle heat, becomes yellow and brownish while dissolving,
and then the purple colour appears without any addition of sugar.
Consequently there is a piind facie presumption that psychosin
still contains the cerebrose, while from sphingosin it is detached.
We have already seen how well this presumption, derived from
the chemical reactions of these bodies, is supported by their
relative chemical formulae. The reaction can consequently be
used for demonstrating the purity or impurity of any specimen of
sphingosin as regards its freedom from or contamination with
substances capable of yielding cerebrose with oil of vitriol. The
substances most likely to remain mixed with sphingosin in
158 CHEMICAL CONSTITUTION OF THE BRAIN.
small quantity are those of which it is a cleavage-product,
and more particularly psychosin, which, like sphingosin, is an
alkaloid.
Isolation of Hie Purple Products. — The purple bodies produced in
the reaction of the cerebrin-products described in the foregoing
pages are, under certain conditions, soluble in chloroform ; it is
necessary to place the mixture in bottles carefully stoppered, and
keep them anhydrous by excess of oil of vitriol. The clear dry
chloroform solution can be distilled from dry vessels boiling, and
leaves the purple product behind. The residue dissolves in new
chloroform with a finer purple colour than before and completely.
A little water added to the purple chloroform solution makes it
turbid, and destroys the colour completely in a few minutes.
The purple, which has been redissolved after the removal of the
first chloroform by distillation, is destroyed by water instan-
taneously.
Caramel of Psychosin. — Experiment. — 0-3615 g. heated at 110°
C, in the apparatus used in the other cases, lost '021 g., and
the CaClg gained -0225 g. At this temperature the substance
became brown and caked. It was then heated to 160° C, when
it fused completely, losing -0145 g., the gain being at the same
time -0185 g. At 210' C. it had lost -021 g., while the CaCl2
had gained the same amount. On cooling, it split up into flakes,
which were only partially soluble in ether. At 210° 0. the
current of air was stopped, as there was a slight deposit of
volatile matter on the cooler parts of the tube. The current was
resumed as the caramel cooled.
Tahular View of the Data concerning the Caramel of Psychosin.
Dry
Amount.
Temp.
Per
cent.
Loss.
Total
per cent.
Loss.
Molecles.
Per
cent.
Gain.
Total
per cent.
Gain.
Molecles.
•3615
110°
5-81
6-22
160°
4-01
5-12
210°
5-81
15-63
4-038
5-81
17-15
4-43 '
Bemarls on the Caramels. — The preliminary experiment on the
action of heat upon phrenosin showed a loss of 10-2 per cent.,
which is equal to 4-03 molecles of water on the formula
C^jHwgNOs. The maximum loss which phrenosin experiences in
the experiments described later is similar in amount, but the
NITROGENISED NONPHOSPHOFJSED PRINCIPLES. 159
water collected does not tally with it. It is therefore clear that
some oxidation takes place in the substance under caramelisation ;
this hypothesis does not, however, completely explain the dis-
crepancies. In future experiments the operation should be
carried on in a current of neutral gas, such as hydrogen or
nitrogen, or in carbonic acid.
/. Intermediate Products of the Chemolysis of the Cerehrosides tvith
Sulphuric Acid: Hydrated Phrenosin, j^sthesin, Psychosin.
The decomposition of the cerehrosides by barita ensues in a
much shorter time than by acids. The products, though essen-
tially the same in both processes, are differently distributed,
and are split off at different times and in a different order. It
is essential to know all those products which are intermediate,
in the first instance, because without them the formulae of the
decomposition cannot be made evident with all necessary detail,
and secondly, because small quantities of these intermediate pro-
ducts mostly outlast the chemolytic process, and then occur as
impurities in the final products or are left as residues incapable
of purification and analysis.
The first event in the sulphuric acid chemolysis of phrenosin is
])voh2ih\y hydration. The next event is t\iQ splitting off of the cere-
brose. This, in the water solution, ensues very slowly, while the
first hydration is probably more quickly attained.
The remainder of the radicles, minus the cerebrose, do yet hold
together for a longer time before they- split up into sphingosin,
fatty acids, and other bodies.
Forty -four g. of mixed cerehrosides were boiled in water, and
pressed through a cloth. To the homogeneous paste 40 cc. of
oil of vitriol, already diluted with the amount of water necessary
to prevent overheating, were added. The mixture was now
boiled, and curdled immediately. Boiling was continued for
nearly an hour, when, the liquid containing much sugar and
becoming more coloured, heat was withdrawn.
The curdled cerebrin matter was transparent and soft while hot,
slightly coloured red, and became solid and white immediately
on cooling. In order to remove any fatty acids which it might
contain, it was, after removal of all sulphuric acid, suspended in
ammonia water, and precipitated by barium chloride. The curdy
160 CHEMICAL CONSTITUTION OF THE BBAIN.
precipitate was found to be almost entirely soluble in hot spirit,
and to contain hardly any fatty salt insoluble in spirit.
The spirit solution filtered hot through a heated funnel imme-
diately deposited white crystals. These, under the microscope,
were seen to consist of two bodies, one in needles, another in
crystalline balls. The deposit from spirit was isolated and sus-
pended in ether.
A hody dissolved which crystallised from the evaporating ether
in apparently curved needles, which will presently be more closely
defined. Another body remained insoluble in ether, and on
combustion left some barita. This latter will not be considered
any further in this place.
Crystallised soluble in Ether Product. — JEsthesin. — The ether
solution was distilled to a small bulk, and then allowed to crys-
tallise. It formed a voluminous white mass consisting entirely
of crystals, which were uniform and showed many angular plates.
They were collected on a filter and drained of mother-liquor by
stirring. While wet they fused on the water-bath in the mother-
liquor adhering to them, but became completely dry without
much discoloration, and were solid and waxy, therefore not
fusible below 90^ Recrystallised slowly from a dilute solution
in ether, the crystals are seen to be hexagonal plates, more or
less regular, but whether or not the six angles of the hexagons
are equal cannot be determined. The plates are saucer-shaped,
and this produces the appearance of the curved or sickle-shaped
needles when the bodies are seen sideways and the lower edge
is out of focus. The plates are scarcely visible when they lie
flat on the glass. They are distinctly recognised with all the
details when they are made to roll edgeways over the field
(Chinese hats).
The dry crystals dissolve in oil of vitriol and assume a yel-
lowish colour, but no purple colour. Cane-sugar added to this
produces the purple reaction soluble in chloroform and yielding
a specific spectrum ; consequently, this body contains the oleo-
cholide radicle, but not the cerebrose radicle. It was now
analysed. Between 80° and 150° 0*1825 g. lost in three stages
5 mgrs. in Aveight; the remainder burnt left 1 mgr. incombustible
residue. The dry powder fritted a little, and at 82° to 83° became
a waxy transparent mass. It fused to a liquid at 110°, but did
not become opaque until again cooled to 71°. By recrystallisation
NITROGENISED NONPHOSPHORISED PRINCIPLES. 161
its nitrogen, which at first was 2*70 per cent., could be depressed
to 2-35. After purification and crystallisation from absolute
alcohol and from pure ether, in both of which it was soluble in
the cold, it crystallised in the same hexagonal plates. Heated to
150° it became coloured, and exhaled an odour of burnt fat. For
analysis it was heated to 100° for three hours, and gave results
which are represented in the following summary :
Percents.
-f-At. Wgts.
-4-N = l.
c
74-83
6-24
37^14
H
12-73
12-73
75-79
N
2-35
•168
1-00
0
10-09
•631
3-75
These results prove that sesthesin is an intermediate product
of chemolysis ; it does not contain the cerebrose radicle, as is
evident from the low quantity of oxigen it contains, and it still
retains the oleo-cholide radicle, as is evident from its yielding the
purple reaction when sugar is added. It therefore behaves like
a sphingosin to which a fatty acid radicle is still combined. The
nitrogen is a little too low ; that is to say, sesthesin is already
mixed with a trace of fatty acid, but the oxigen stands to carbon
in the relation of about 3 atoms to 35 atoms. Hence the hypo-
thesis that sesthesin is a compound of sphingosin and neurostearic
acid gains a high degree of probability, thus :
Sphingosin. + Neurostearic acid. JEsthesin. Water,
G^,'^, + G^^, = C^coNO^ + H,0.
If now to sesthesin we add a molecle of" cerebrose, we obtain —
^sthesin. Cerebrose. Phrenosin. Water.
The fatty acid radicle contained in sesthesin is supposed to be
neurostearic acid.
Theory.
Pound.
Theory of a
Hydrate.
Atoms.
Percents.
Percents.
Atoms.
Percents.
35 C 420
76-22
74-83
35 C 420
73-81
69 H 69
12-52
12-73
71 H 71
12-47
1 N 14
2-72
2-35
1 N 14
2-46
3 0 48
8-54
10-09
4 0 64
11-26
551
100-00
569
11
162 CHEMICAL CONSTITUTION OF THE BRAIN.
The aesthesin analysed did, therefore, yet contain about half a
molecle of water. The body seems to be a base.
g. Chemolysis of Fhrenosin hy Sulphuric Acid in Alcoholic Solution:
Formation of Ethylic Neurostearate.
The Process. — Sixty-five g. phrenosin were suspended in 1500 cc.
of spirit of 85 per cent, strength, and 200 cc. of oil of vitriol
gradually added while the mixture was well agitated. The hot
mixture, on which an oily matter floated, was then boiled for
two hours and a half in such a manner that the volatilised alcohol
was condensed and ran back again into the fluid. On cooling, the
oily layer solidified, and a slight deposit formed in the underlying
fluid. The insoluble and deposited parts were separated by filtra-
tion from the acid solution and dissolved in ether ; the ethereal
solution was agitated with dilute solution of caustic soda, by which
a small quantity of fatty acid was removed as soda salt. The
clarified ether solution was distilled, and the residue was re-
peatedly crystallised from absolute alcohol. From this solvent it
was deposited in small shining crystals, which were very soluble
in hot alcohol, but very little in cold. The recrystallised matter
fused at 56° C, but the manner of its fusion seemed to indicate
an admixture of a less fusil)le with a more fusible body. It was
subjected to a preliminary elementary analysis by the vacuum
method with the following result : 0 0872 g. gave 0-1080 g.
HoO, 0-2443 g. CO.,, and 0-00043 g. nitrogen. The small amount
of nitrogen was evidently due to the presence of a small quantity
of a nitrogenised substance which was at once eliminated by the
process of distillation in vacuo about to be described. The re-
crystallised body was mixed with glass powder and placed in a
glass tube closed at one end. This was then bent dov/nwards at
a point beyond the mixture, and a little further on bent again
upwards, drawn out and connected air-tight to a mercurial air-
pump. The air was then completely pumped out, and heat
cautiously applied until the matter was distilled into the V-shaped
part of the tube ; this was effected without the evolution of any
gas and without a trace of charring. When at the end of the
distillation the slight residue in the glass powder began to discolour
and to evolve gas, the operation was stopped.
Ncurostearic Ether, C2oH^^02. — The distilled matter was of the
colour and consistence of bleached beeswax. It melted at 52° C.
NITROGENISED NONPHOSPHORISED PBINCIPLES. 163
On analysis it gave numbers agreeing with those demanded by the
formula C^qH^qO^. It contained no trace of nitrogen.
Theory. Found.
Atoms. Percents.
240 76-93 76-69
40 12-82 12-95
O, 32 10-26 10-36
312 100-00 10000
The body is ethylic neurostearate {Q.2^r)Q-^^.^fi.2^ as was
proved by the following experiment : 4*5 g. of the ether, which
had been completely freed from any adhering alcohol by fusion at
110° during several days, were enclosed in the platinum chemo-
lyser with concentrated soda ley, and heated to 100° for eight
hours, during which the chemolyser was frequently shaken. The
caustic solution when quite cold was poured upon a filter of glass
wool, and by this means the soap, which was quite insoluble in
the concentrated ley, was separated from the excess of- alkali ;
and, after rinsing with cold, was dissolved in a large quantity of
boiling water. It gave a clear solution. A portion of this solu-
tion, precipitated hot with barium chloride, gave a barium salt.
This was converted into free acid, which was dried and dissolved
in ether. On concentration, the ether gave a crystallisation of
perfectly white neurostearic acid in a characteristic form. The
ethereal mother-liquor crystallised similarl}^ in cauliflower masses
to the last drop. This acid fused at 84° C. It was dried by
fusion at 100° C, and analysed with the following results :
Theory. Found.
Atoms. At. Wgts. Percents. Percents.
216 76-06 75-94
Hg^ 36 12-68 12-64
Oo 32 11-26 11-42
284 100-00 100-00
The concentrated soda ley employed in this chemolysis of the
neurostearic "ether was examined for alcohol. It was distilled, the
distillate enclosed hermetically in a glass tube with excess of
chromic and sulphuric acids, and heated for six hours to 100°.
The chromic acid was partially reduced, and on distillation of the
fluid an acid distillate was obtained. This, after boiling with
11—2
164 CHEMICAL CONSTITUTION OF THE BBAIN
barium carbonate, and evaporation to dryness, gave 0-32 g. dry
barium acetate, giving a red coloration Avith ferric chloride, evolv-
ing the pungent fumes of acetic acid when moistened with oil of
vitriol, and giving the fragrant odour of acetic ether with alcohol
and oil of vitriol.
Psychosin Sulphate. — The alcoholic mixture of sulphuric acid
and other decomposition products from which the neurostearic
ether had been removed by filtration, was treated with caustic
lime in powder, and the liquid, now free from sulphuric acid,
filtered from the gypsum and some calcium neurostearate which
was mixed with the precipitate. The alcoholic solution was dis-
tilled, and, after the alcohol had passed off, left a voluminous
white pasty mass of free psychosin. This was dissolved in hydro-
chloric acid and ether, and thus separated from the impurities, of
which sulphovinate of calcium was the principal one. The ether
solution of psychosin hydrochlorate was freed from ether by dis-
tillation, the residual salt dissolved in water, and allowed to stand
in the . cold. It deposited colourless crystalline plates, masses of
fine microscopic crystals of extreme tenuity, but showing geometric
definition of sides and. angles; these easily redissolved if the
liquid was allowed to rise in temperature. Filtration was there-
fore effected in the cold. These crystals consisted of pure sj^hin-
gosin hydrochlorate, as was proved by isolation, decomposition
with caustic potash in the hot, extraction of the base with ether,
solution of the base, after distillation of the ether, in absolute
alcohol, and precipitation of the sulphate by oil of vitriol dissolved
in absolute alcohol. The psychosin in the solution was identified
by isolation and all the characteristic tests.
A. Action of Heat upon Phrenosin ; Formation of a Caramel.
Freliminary Experiments. — Some preliminary experiments for
the study of the action of heat upon phrenosin may be here again
referred to (see 'Eeports,' etc., New Series, No. III., 1874, p. 191).
'0-5290 g. lost, on drying between 18° and 70° C, 0'0084 hygro-
scopic water, and the remaining 0-520G g. was considered as dry
substance. After two hours' exposure to a heat of 97° C. in an
air-bath it became slightly yellow, and had lost 0-0016 g. After
three hours' exposure to 101° it had remained of the same colour
and weight. After two hours' exposure to 1 45° it had become
NITROGENISED NONPHOSFHORISED PRINCIPLES. 165
very dark, almost black in colour, and superficially fused, and had
lost 0*0080 g. After heating to 158° during three hours it had
become thoroughly fused, and of a reddish, almost transparent
aspect ; it had lost 0*0120 g. in weight. After four hours at
177° 0. it had become blacker and less transparent, and gave out
a faint odour of burnt meat : it had lost at this stage 0*0317 g.
Thus the phrenosin had lost in four stages 0*0533 g., or 10*2 per
cent, in weight. After cooling, it was hard and brittle. On
boiling a piece in absolute alcohol, only a trifling amount of
matter dissolved, colouring the solution slighty yellow. On the
other hand, it readily dissolved in ether ; the solution had a dark
reddish-brown colour. A little of the phrenosin was heated in a
test-tube over the naked flame. It fused, turned dark, and
evolved water with ebullition, similar to sugar passing into
caramel. The water, which condensed in the upper part of the
tube, had an acid reaction, and reduced copper solution. The
fused matter became hard on cooling, was but slightly soluble in
boiling alcohol, but readily soluble in ether ; from the latter
solution it was reprecipitated by alcohol.'
Similar exj^eriments were now instituted, with a view of in-
creasing the accuracy of results and the knowledge concerning
the constitution and function of phrenosin.
First Experiment. — Some phrenosin was dried in a current of
air at 100° C, in a Liebig's drying-tube placed in a paraffin bath,
connected at each end with a chloride of calcium drying-tube, one
to dry the air before it entered the tube, and the other to arrest
the moisture from the phrenosin evolved during the operation.
A drying-tube filled with glass saturated with sulphuric acid
served to prevent moisture from the water-pump diffusing back-
wards into the chloride of calcium tube.
The dry phrenosin weighed 1*216 g. The temperature was
raised to 150° C, a current of dry air passing meanwhile. When
the tube containing the phrenosin was weighed, it was found to
be still of the same weight as at the beginning of the experiment ;
but the chloride of calcium tube had gained *019 g., a fact which
can only be explained by oxidation of the phrenosin. At this
stage the phrenosin became brownish-white in colour, but did not
fuse. The temperature being now raised to 160°, the phrenosin
melted into a brown mass, and on being weighed was found to.
106 CHEMICAL CONSTITUTION OF THE BRAIN.
have lost -014 g., the chloride of calcium tube having gained the
same amount. The temperature was then rapidly raised to
200° C, but there was no longer a current of air allowed to pass
through, in order to retard as much as j^ossible the evolution of
volatile matter. On cooling, air was passed through as before,
and the tube on being weighed showed a loss of 0 054 g., the
chloride of calcium tube having only gained 0*050 g. The tem-
perature was now finally increased to 240' C, with the same
precautions against escape of volatile matter as before. Volatile
matters were now, however, evolved which slightly blackened the
sulphuric acid ; and when the connections of the apparatus were
severed, a smell of burnt sugar was perceived. The tube showed
a loss of 0*080 g., while the chloride of calcium tube had gained
0*056 g.
Thus in the whole operation the tube had lost 12*17 per cent,,
while the chloride of calcium had gained 11*425 per cent. Taking
the formula of phrenosin as C^^HyglSTOg, the molecular weight of
it would, be 713. The preceding figures would then give us,
taking the loss as 12*17 per cent., a loss of 4*82 molecles of
water; and, taking the gain as 11*425 per cent, a loss of 4*52
molecles.
Second Experiment. — Some phrenosin was dried at 100° C, the
weight of the dry body being 0*789 g. The temperature of the
tube was now raised to 200° C, and kept at that point for two
hours, air being passed through only on cooling below 150'. No
volatile matters were carried into the sulphuric acid tube, though
a slight sublimation of volatile matter on to the cooler parts of
the tube took place. On weighing, the tube was found to have
lost 0*0456 g., and the chloride of calcium had gained 0*054 g.
The heating was now continued at 200° for four hours, to try the
effect of time on the operation, air being passed through only on
cooling. The loss was now 0*0365, and the gain 0*0365. The
totals in percents. were a loss on the part of phrenosin of 10*14
per cent, or 4 02 molecles, and a gain on the part of the calcium
chloride of 11*47 per cent, or 4*54 molecles of water.
The greater portion of the caramel obtained in both these ex-
periments was soluble in ether; the first caramel was not so
perfectly soluble as the second, on account of the greater tem-
perature to which the former had been raised.
NITROGENISED NONPHOSPHOEISED PRINCIPLES. 167
Tabular View of the Data concerning Caramel of Phrenosin.
I^^y T^rv^r. '^""^""K A/r 1 1 Percent. Total Molecles
Amount. ^^^P- Loss, per cent. Molecles. (.^i^. Pei" cent. jj q_
Gain.
1-216 1.50 -00 — — 1-56 — —
— 160 M5 — — 1-15 — —
200 4-44 — — 4-11 — —
— 240 6-58 12-17 4-82 4-605 11-425 4-52
-789 200 5-89 — — 6-85 — —
— 200 4-25 10-14 5-02 4-62 11-47 4-54
i. Special Reactions of Phrenosin.
Reaction of Phrenosin with Oil of Vitriol^ Chloroform and Glacial
Acetic Acid; Spectral Phenomena of the Product. — Phrenosin from
the brain of man was suspended in chloroform, and sulphuric acid
added ; this was allowed to stand five minutes, and the chloroform
was then decanted ; it remained white and clear. On being
allowed to stand over the sulphuric acid all night the chloro-
form was still colourless, but the sulphuric acid was darker ; the
latter was dissolved in glacial acetic acid with a little sulphuric
acid in order to dissolve some red particles. The solution so
produced had a magnificent red colour, and presented spectra as
follows : In a concentrated solution red only passed. This
solution had a green fluorescence. More diluted three bands
appeared, one between D and E, a second between E and F, and
a third between F and Gr.
Reaction of Phrenosin with Pettenkofer's Test ; and Spectrum of its
Product. — Phrenosin from the brain of man was dissolved in boiling
chloroform ; it became turbid on cooling, but did not become con-
gealed. This solution was now mixed with sulphuric acid and
sugar, then stirred and repeatedly breathed upon ; it formed
purple oily drops, but no solution took place. These drops were
insoluble in glacial acetic acid, but dissolved rapidly in chloro-
form. The solution, kept anhydrous by sulphuric acid, presented
the following spectrum with Drummond's light : a narrow band
between C and D, and a wide deep black band between D and h.
These reactions of phrenosin are very similar to those of kerasin
to be described. They are also very similar to those of oleic
acid.
168 CHEMICAL CONSTITUTION OF THE BRAIN
k. Theory of the Chemical Constitution of Phrenosin.
Although the average of the analyses of phrenosin leads to data
from which the true formula of its composition can be derived, it
is a great advantage to be able to prove the true formula by the
aid of the formulae of the products of decomposition. If phrenosin
had never been analysed by itself, its formula could be predicted
by means of the synthesis of its cleavage-products, according to
the followinsf calculation :
1 molecle of sphingosin - . . Cj^Hg^NO^.
+ 1 ,, of neurostearic acid - - C^gHg^ O^.
+ 1 „ of cerebrose - - " H^^ ^o-
— 2 molecles of water - - - - 0.,.
leave one molecle of phrenosin - C^^H-gNOg.
Arranged analytically the equation would be the following :
C„H,,NOs + 2(H,0) = C,^,,m, + C,,U,fi, + C,U,.fi,.
Of this phrenosin the atomic and percentic theory is the follow
Theory. Found mean.
^ ' ,
Atoms. Percents. Percents.
41 C 492 69-000 67 957
79 H 79 11-080 11-426
1 N 14 1-963 1-997
8 0 128 17-957 18-696
mo; :
713 100 000
This phrenosin, in the course of chemolysis, by taking up two
molecles of water, increases its atomic weight from 713 to 749,
which latter figure represents the sum of the atomic weights of
the three products of decomposition above enumerated. 100
parts of phrenosin, therefore, become 105 parts of products. Of
these 39-6 parts are neurostearic acid, 39-9 parts sphingosin, and
25*1 parts cerebrose.
If now, with the aid of this theory, we attempt to revise the
theories formerly given of some compounds of phrenosin, re-
taining the analytical data, we obtain a greater harmony than
before. Thus the nitrited phrenosin shows the following theory :
Nitrited Phrenosin Nitrate, C^iH^y(NO^,)NOg -f HNO..3, or con-
tracted, C^lH^gNyOiy
NITROGENISED NONPHOSPHORISED PllINCIPLES, 169
Theory.
Atoms.
41 C 492
79 H 79
3 N 42
13 0 208
Percents.
59-92
9-62
5-11
25-33
Found
in 100.
59-58
9-93
4-65
25-84
821
Neurostearic Acid is accurately distinguished from the isomeric
known common stearic acid by its high fusing-point (84° to 85^"),
and its atomic weight is well fixed by its ethyl compound. But
its salts are not very stable nor very precise bodies, or are
mixtures, so that it has hitherto proved impossible to use them for
stoichiometric purposes. The theory of this acid is the following :
Theory. Found
Atoms.
18 C 216
36 H 36
2 0 32
284
Percents.
76-056
12-676
11-268
100-000
in Crystallised Acid.
75-88
12-85
11-27
in Acid from Ether,
75-94
12-64
11-42
100 00
100-00
The ethylic compound of this acid is a very precise body ; it is
Neurostearic Ether or ethylic neurostearate, and its theory is the
following
Theory.
Found.
r
Atoms.
Percents.
Percents.
20 C 240
76-92
76-69
40 H 40
12-82 •
12-95
2 0 32
10-26
10-36
312
100-00
100-00
The dissolved formula is
Sphingosin is a strong base, and gives the most precise compounds
of any products of the decomposition of phrenosin. Its theory is
the following :
Theory.
Atoms.
17 C 204
35 H 35
IN 14
2 0 32
Percents.
71-59
12-28
4-91
11-22
Percents. found
in Sulphate,
Acid deducted.
71-26
12-53
4-82
11-39
285
100-00
10000
170 GHEMICAL CONSTITUTION OF THE BRAIN.
Sphingosin Sulphate, 2(Ci^H35NO^) + H2SO4.
Theory.
Found.
Atoms.
Percents.
Percent s
34 C 408
60-11
60-85
72 H 72
10-78
10-70
2 N 28
4-19
4-14
4 0 64
10-55
9-99
IS 32 )
4 0 64 1
14-37
14-32
668
100-00 .
100-00
Cerehrose, CgH^^Ojj, is a sugar characterised by its crystallisation,
its optical power (its specific or limited rotation being + 70° 40'),
and its reducing power over cupro-potassic tartrate. Its theory
is the following :
Theory.
Found.
Atoms.
Percents.
Percents.
6
C 72
40-000
39-93
12
H 12
6-666
6-71
6
0 96
53-334
53-36
180
100-000
100-00
Under certain conditions which have been described above,
cerebrose is changed into an acid isomeric with cerebrose, and
therefore of the formula C^H^^O,^. This cerehrosic acid is dibasic,
i.e.f contains two atoms of hydrogen replaceable by metals. The
theory of the cerebrosate of barium is the following :
Theory. Found.
Atoms. Percents. Percents.
6 C 72 22-85 24-53
10 H 10 3-17 3-2
1 Ba 137 43-49 43 -5
6 0 96 30-49
315
This acid is remarkable in this, that it has not got any reducing
power over potassio-cupric tartrate, but on the other hand gives
with an oleo-cholide radicle, e.g. sphingosin, and oil of vitriol the
purple reaction in the same manner as cerebrose.
Psychosin, C.yfl^r^'NOj, is the cerebroside of sphingosin ; it is
crystallisable from alcohol ; it is an alkaloid, but of less pro-
NITROCtENISED NONPHOSPHORISED principles. Vil
nounced character than sphingosin ; it forms salts with acids, which
are more or less soluble in water, the hydrochlorate being very
soluble indeed. By this solubility in cold water it can be separated
almost completely from the hydrochlorate of sphingosin, which
crystallises from cold water, or cold solution of psychosin hydro-
chlorate.
Found.
Theory Percents.
Atoms.
Percents.
h.
23
c
276
61-74
61-86
61-32
45
H
45
1006
10-09
10-09
1
N
14
3-13
2-88
3-04
7
0
112
25-07
25-17
25-55
447
10000
One hundred parts of psychosin on chemolysis should take up
4-02 parts of water (one molecle), and then split up into 40-29
parts of cerebrose and 63-75 parts of sphingosin.
The hydrochlorate is completely precipitated by excess of
hydrochloric acid. Many compounds may be expected to be
obtained, as psychosin exhibits numerous promising reactions.
It gives the oleo-cholide reaction with oil of vitriol alone, showing
that it contains the cerebrose and sphingosin radicles.
The Caramels of the cerebrosides are, like the caramels of the
sugars, produced by the expulsion of water under the influence of
heat. They are all soluble in ether, insoluble in alcohol and in
water, and of a deep brown colour. The following formulae are
hypothetical and interimistic, although, derived from the data of
the experiments given above.
Caramel of Phrenosin, C^Ji>j^O^, formed from a molecle of
phrenosin, C^^H^gNOg, by the loss of four molecles of H^O.
Caramel of Psychosin, C^gHg^NOg, formed from a molecle of psy-
chosin, by the loss of four molecles of water.
A small quantity of caramel is formed during every chemolysis
of any of the cerebroside principles, with acid or with alkali, even
during the chemolysis of psychosin with dilute sulphuric acid.
There may be several varieties of caramel of each principle pro-
duced by the loss of one, two, three, or four molecles of water.
There might be mixed with some preparations of phrenosin parti-
cular varieties of phrenosin, in very small quantity, containing in
place of the neurostearic another fatty acid radicle. And there
172 CHEMICAL CONSTITUTION OF THE BRAIN.
might be mixed with an hypothetical di-neurostearyl-sphingosyl-
cerebroside, a similarly constituted body containing in place of
one or both molecles of neiirostearyl other fatty acid radicles, and
in place of the sphingosyl another nitrogenised radicle ; in this
respect the nitrogenised principles might imitate the phosphoriscd
principles, which derive their principal differences from the dif-
ferent fatty acid radicles which they contain. I think it even
very probable that some of the phosphoriscd bodies which cling
so pertinaciously to the nitrogenised ones owe their similarity to
these latter, both in shape and chemical properties, to the fact of
their containing one or other or both of the radicles of the nitro-
genised bodies which appear as neurostearic acid and sphingosin.
3. Kerasin, the Second Cerebroside: its Isolation and
Properties.
Tntrodudion. — As in the case of phrenosin. I have, up to the
present moment, not succeeded in isolating a preparation of
kerasin which, on analysis of a sufficient quantity, proved to be
free from phosphorus. The preparation which yielded the lowest
amount of phosphorus, namel}^ 0-08 per cent. (8 parts of phos-
phorus in 10,000 parts of kerasin), weighed only 4 g., so that
after six quantations, for which the materials were taken from
these 4 g., there was not left material enough to be employed in
a systematic attempt at chemolysis of this particular specimen.
But I have examined a considerable number of preparations of
kerasin which contained more of the phosphoriscd impurity, not
exceeding 0-4 per cent, of P ; and some of these specimens have
yielded information which, when compared with the information
derived from the purest specimens, seemed to be independent of the
influence of the thus far unavoidable impurity. But the informa-
tion, though decisive as far as it goes, is only fragmentary as
regards the entire problem ; and I therefore point out that what
I have to report is only the beginning of a great research to be
made in the future.
Mode of Isolation. — The ox white matter, which had been ex-
tracted with ether and dried, was pulverised and dissolved in hot
absolute alcohol ; the solution was decanted from the fused mass
of stearoconot which formed, and allowed to deposit the dissolved
matter by cooling. The deposit formed after the first hour was
isolated, as was also another which formed after the second hour ;
NITROGENISED NONPEOSPHORISED PBINCIPLES. 173
a third gelatinous-looking precipitate formed over-night, and from
this the absolute alcohol solution was filtered. On standing for a
few days in stoppered bottles, this solution deposited a gelatinous
membranous mass, mainly consisting of Jcerasin. This was re-
moved by the filter, and the filtrate, which contained much
sphingomyelin in solution, was treated with CdCl^. The white
bulky precipitate of sphingomyelin CdCl2 was filtered off, washed,
exhausted with ether, and further treated as is described else-
where. It may be mentioned here that the principal bulk of
sphingomyelin is obtained in this manner and at this stage. The
alcohol filtered from the sphingomyelin CdCl2 precipitate after
concentration deposited a mixture of CdCl2 salt, cholesterin,
and kerasin, the latter two being present in very small pro-
portions. ,
Mode of Purification. — Kerasin obtained as above is a soft white
gelatinous mass, consisting of larger and smaller balls, which
under the microscope are seen uniformly to consist of wavy
masses of needles so thin that it may be said they possess only
one diameter— namely, length. The gelatinous state is apparently
entirely due to this peculiarity of the fine needles enclosing a
large amount of alcohol. No amorphous matter whatever is seen
mixed with it, but here and there a few rosettes of phrenosin,
strikingly differentiated from the kerasin.
The whole of the kerasin was dissolved three times in boiling
absolute alcohol, and, after cooling to crystallisation, washed and
pressed free from mother-liquor. This, after the third operation,
was free from sphingomyelin, as shown by the absence of reaction
with PtCl^, and CdCl^. The solution of kerasin was also free
from sphingomyelin, CdCl^ giving no precipitate with it.
The kerasin was now dissolved in a fourth quantity of pure
absolute alcohol, and allowed to crystallise. It was found by
microscopic examination that after three hours much kerasin in
wavy crystallised masses, but no phrenosin in rosettes, had been
deposited. The crystals were consequently isolated, pressed,
again recrystallised, collected on a filter, and dried in vacuo.
This preparation was analysed with the result stated below.
After this preparation had been removed, at the end of the
third hour, from the mother-liquor, the latter on standing de-
posited a mixture of much kerasin in wavy needles, with some
rosettes of phrenosin. This mixture could by recrystallisation
174 CHEMICAL CONSTITUTION OF THE BRAIN
not be completely separated. A third ultimate precipitate seemed
to consist mainly of phrenosin, with much kerasin, which could
also not be purified hj mere recrystallisation.
Special Consideration of the Properties of Kerasin tvhich are r)iade
use of for Its Isolation. — The properties of kerasin which are made
use of for its isolation are the following : It is easily soluble in
hot spirit, and almost insoluble in cold ; it tarries to deposit from
this solution for a long time, and if its amount does not exceed
1 part in 321 parts of spirit, it is not deposited at all above 28°,
and below that temj)erature very slowly. By this peculiarity it
is separated from phrenosin and sphingomyelin, the latter parti-
cularly when it is in the state of cadmium chloride salt. Kerasin
does not combine with lead ; by this property it is separated from
myelin and the cerebrinacides, which combine with lead. It does
not combine with cadmium chloride, and by this reagent is
liberated from sphingomyelin, amidomyelin, and paramyelin,
which combine with it. It is not soluble in ether, either cold or
boiling ; by cold ether the kephalins and lecithins are extracted
from it, and by boiling ether, krinosin. Kerasin swells when left
in contact with ether for some time. In that state it must not be
allowed to dry on paper, as it contracts and becomes hard, and
adheres so strongly to the paper that it cannot be separated from
it without retaining fragments of paper in its substance. Kerasin,
crystallised from absolute alcohol in a flask, if allowed to dry
slowly in the flask after the alcohol has been poured off, becomes
white and pulverulent, so as to crumble off the sides of the glass,
and does hardly become waxy. When deposited from watery
solvents, or removed from contact with even anhydrous ether, it
becomes on drying hard and horny, or waxy, and difficult to
powder.
Solubility of Kerasin in Different Quantities of Spirit. — 3-8878 g.
human kerasin, dried on the water-bath, we^e dissolved in 500 cc.
hot spirit of 84 per cent, strength. On cooling of the solution a
deposit of kerasin began to form at 40°, and continued to increase
while the temperature sank. 200 cc. spirit were added to the
mixture, and the deposit was redissolved by the application of
heat. On being again allowed to become cool, it now became
turbid, and began to make a deposit at 35^ A further addition
of 50 cc. of s})irit reduced the depositing-point to 34°. Another
100 cc. reduced the crystallising-point to 33°. A further 100 cc.
NITROGENISED NONPHOSPHORISED PRINCIPLES. 175
depressed the temperature of crystallisation to 32°. Three more
additions of 100 cc. spirit each depressed the crystallising-point
to 30% 29°, and 28° respectively. Altogether, therefore, 3-8878 g.
kerasin required 1250 cc. of spirit to be kept in solution at 28' ;
one part of kerasin required 321 parts of spirit to remain in solu-
tion at 28°.
Fifty cc. of a solution of kerasin in spirit, which began to
crystallise at 28°, were at that temperature filtered and evaporated
to dryness on the water-bath. The residue weighed 0*1558 g. ;
therefore 1 g. kerasin required 320*92 cc. of spirit at 28° for
solution.
Solubility of Kerasin in Aceton. — At the ordinary temperature
of the air, 100 cc. of aceton dissolve 0*1576 g. kerasin; at the
boiling temperature of aceton, 100 cc. dissolve 1*0510 g. kerasin.
Solubility of Kerasin in Benzol. — When moist kerasin is shaken
with large volumes of benzol, it remains as an insoluble gelatinous
transparent mass. The filtered benzol leaves no residue on dis-
tillation. When kerasin and benzol are warmed, a perfect solution
is produced, which can be filtered hot. On cooling, all kerasin is
deposited, and the benzol leaves no residue on distillation. This
solvent, therefore, may serve for the separation of bodies which
are soluble in cold benzol.
Reactions of Kerasin. — Both human and bovine kerasin give a
deep purple with oil of vitriol and sugar at once ; with oil of
vitriol alone, a paler purple after long standing. Kerasin from
ox-brains is dissolved in boiling chloroform. On cooling, it con-
geals to a glassy solid. A portion of this is stirred with a drop of
cane-sugar solution and a small quantity of sulphuric acid. At
first it becomes yellowish, and at last purple. The colour is in
drops, and not dissolved ; it is not soluble in glacial acetic acid,
but chloroform dissolves the whole to a purple solution. This
before the spectroscope shows a narrow band between C and D,
and a deep black band extending from D to F. The acid solu-
tion below the chloroform, is yellowish, fluorescing green. When
some kerasin is dissolved in chloroform, and sulphuric acid
added, all the kerasin passes into the acid, and the chloroform
remains colourless : this proves that the kerasin is free from
cholesterin. When this mixture is allowed to stand, a brownish-
red mass rises to the top of the acid ; sugar added to this makes
it redder. W^hen this is placed in a dish and the chloroform
176 CHEMICAL CONSTITUTION OF THE BRAIN
evaporated, oily deposits form of increased redness, the oxigen of
the air evidently tending to make them purple ; but this purple
product is now insoluble both in acetic acid and chloroform, singly
or united.
Kerasin, in a Liebig's drying-tube, heated in an oil-bath to
from 100° to 150° in a current of dry air, loses about 4 molecles of
water, and is transformed into a brown matter soluble in ether,
insoluble in alcohol (caramel of kerasin).
Elementary Analyses and Theory of Kerasin.— A. specimen of
kerasin from the ox obtained from 70 g. of the principle by fre-
quent fractional .recrystallisation, and containing yet 0*01 per
cent. P, was analysed, and yielded the first group of data in the
synopsis below.
A specimen of human kerasin was repeatedly crystallised from
absolute alcohol, allowed to dry, and crumble in flask, and dried
over oil of vitriol in vacuo. It contained 0 073 per cent. P; it
gave on analysis the second set of data in the synopsis.
If we group these analyses so as to let the highest nitrogen
come first, we obtain the following hypotheses :
Synopsis of Analyses of Kerasin from Ox.
Percents.
-^ At. Wgt.
C 69-54
5-795
42-29
H 11-69
11-690
85-32
N 1-92
0-137
1-00
0 16-85
1-053
7-68
100-00
Synopsis of Analyses of Kerasin
from Man.
Percents. -t-
At. Wgt.
~ N=l.
C 69-01
5-75
44-23
H 11-44
11-44
88-00
N 1 -90
013
1-00
0 17-65
1-10
8-46
100-00
Synopsis of Analyses of Kerasin from Ox, made in 1874.
Percents.
-i- At. Wgt.
N =
c
68-446
5-704
46-0
H
J 1-395
11-395
92-0
N
1-738
0-124
1-0
0
18-421
M51
9-2
NITROQENISED NONPHOSPHORISED PRINCIPLES. Ill
The two first sets of analyses are probably to be preferred, and
it is very probable that the formula of kerasin is C42Hg5N03, or
C^^HggNOg ; any formula giving oxygen as eight atoms has theory
in its favour, as we shall see below. But in any case the formula
cannot be established by analysis alone ; chemolyses of kerasin
and analyses of the cleavage products are required to enable us
to form a final opinion as regards its chemical constitution.
Chemolysis of Kerasin. — Kerasin is a cerebroside, and yields
cerebrose by chemolysis with dilute sulphuric acid. In some
chemolyses with barita, the study of the acid products of the
chemolysis could not be accomplished for want of quantity.
But the basic parts were identified in two forms, and the presence
of cerebrose w^as incidentally confirmed.
Four g. of kerasin were mixed with 8 g. of barita hydrate
crystals, and sufficient water to produce a thin paste. The
mixture was enclosed in a tube and heated during fourteen hours
to 100°. The watery solution, free from barita, gave a reaction
for cerebrose. The insoluble in water part was extracted with
alcohol ; the concentrated solution yielded a precipitate of a sul-
phate. Another part of the matter was not precipitated by sul-
phuric acid, but remained in solution. It was shown to be
psychosin, like the base of the precipitated sulphate. This sul-
phate dried to a horny mass on the filter, and did not remain
pulverulent like sphingosin sulphate. It was dissolved in boiling
water, with which it formed a clear solution, and precipitated
with a large excess of caustic alkali. It did not rise to the top
like an oil, as sphingosin does, on heat being applied, but formed
a jelly or semi-soap ; with more potash it became emulged ; the
solution became turbid on cooling ; ether extracted nothing from
it. The only way to extract the alkaloid was to acidify the solu-
tion with sulphuric acid, and add phospliomolybdic acid to it.
The precipitate, after decomposition with barita, yielded to spirit
the psychosin, which was transformed into sulphate and analysed.
Analyses of the Sulphate obtained, and Comparison luith the Com-
position of Psychosin Sulijhate and Sphingosin Sidphate.
Found in Product from Psychosin Sulphate. Sphinsfosin Sulphate.
Kerasin. 2(Co3H4.5N07)H2SOj. 2(Ci7H35NO.,)H2S04.
C 54-67 " 55-65 " 60-11
H 10-48 9-27 10 78
SO^ 10-59 9-67 14-37
12
178 CHEMICAL CONSTITUTION OF THE BE A IN
As the sulphate yields Raspail's reaction without sugar being
added, it must be psychosin, mixed, however, with a small trace
of sphingosin.
It follows from the foregoing that kerasic, like its principal
companion phrenosin, is a cerebroside, namely, a body which con
tains the sugar cerehrose, combined with at least two other radicles.
Of these, one is probably sphingosin, the alkaloid obtained from
phrenosin. The other is certainly a fatty acid, but the nature and
composition of this acid have not yet been perfectly ascertained.
B. SUBGROUP OF THE CEREBRINACIDES, OR CEREBRIN
BODIES WHICH COMBINE WITH METALLIC OXIDES.
1. General Observations on the Subgroup.
The following account of preliminary observations may serve
as the basis for future more perfect inquiries. Of some bodies
the mere existence has been ascertained, and they have not been
definitively isolated. Others have been isolated in a state of
artificial combination Avith reagents. Two have been isolated in
such a state of semi-crystallisation (spherocrystals and groups of
microscopic needles) that their appearance and behaviour give
some probability to the assumption that they are approximately
pure. But the only final control of actual composition, quanta-
tion of atomic weight by combination and demonstration of con-
stitution by chemolysis could not yet be applied to them.
Separation of these Substances from the Cerebrin Mixture by Lead
Acetate and Ammonia. — This process has already been described,
but may here again be noticed. The cerebin mixture, exhausted
with ether, is dissolved in hot spirit, and to the solution a hot
solution in spirit of acetate of lead is added as long as a precipi-
tate is produced. Ammonia is then added, and when neither
lead acetate nor ammonia produce any further precipitate, the
solution is filtered hot from the precipitate. The latter contains
the bodies here to be considered, while the spirit solution con-
tains the cerebrosides and other substances which do not combine
with lead under these circumstances.
The Lead Salts. — The insoluble precipitate formed during the
purification of phrenosin by the lead acetate treatment is ex-
hausted with hot 85 per cent, spirit to remove all phrenosin and
-^^ aCiCOLOrMEDIGlWE
NITEOGENISED NONPHOSPHOBISED PRINCIPLES. 179
kerasin. It is then dried, powdered, and extracted with cold
benzol. A portion of the lead salts dissolved, another remained
insoluble.
2. Cerebrinic Acid : its Isolation and Properties.
The benzol extracts were evaporated to dryness, and the dr}^
residue was powdered and suspended in 85 per cent, spirit. The
spirit was saturated with hydrothion while being gradually raised
to the boiling-point, and the lead sulphide filtered off. On cooling
a white body was deposited, which consisted mainly of cerebrinic
acid. This was collected on a filter, purified by recrystallisation
from absolute alcohol, and dried in vacuo. It gave a weak purple
reaction with oil of vitriol alone. Under the microscope it ap-
peared to consist of small needles. It was soluble in hot benzol,
and was thrown down on cooling as a gelatinous mass. It did
not blacken at 100° C. On elementary analysis it gave data
which are collated in the following table :
a. b. c. d.
Nitrogen per cent. 1-5 — — 1-68
Carbon, per cent. — 66 '47 67*54 —
Hydrogen, per cent. — 11-355 11 '37 —
Caramel of Cerebrinic Acid — First Experiment. — 1*613 g. of dry
cerebrinic acid were heated to 210° C, in a current of air for one
hour. The tube lost 0*122 g., or 7*55 per cent. ; and the chloride
of calcium gained 0*103 g,, or 6*38 per cent. Calculated on a
formula (hypothetical) of C^gH^^gNOg, and a molecular weight of
979, these figures would give a loss of 4*1 molecles of water, as
judged by the diminution of the weight of the cerebrinic acid,
and they would correspond to 3*47 molecles of H2O if only the
gain of weight of the water- catching tube were taken into con-
" sideration.
Second Experiment. — 1*221 g. of dry cerebrinic acid was heated
to 160° C. The substance melted completely, and lost 0*030 g.,
the chloride of calcium gaining 0*033 g. The temperature was
now raised to 200° C, and the current of air was passed only
when the tube cooled. The loss and gain respectively were 0*065 g.
and 0*056 g. The total loss was thus 7*77 per cent., or 4*227
molecles; and the gain was 7*30 per cent., or 3*97 molecles of
water.
12—2
180 CHEMICAL CONSTITUTION OF THE BBAIN
Third Exjjenmeni— 1-073 g. at 150^ C. lost 0-012 g., and the
chloride of calcium tube gained 0*019 g. At this temperature
the cerebrinic acid melted. At 200° C. it lost 0*0965 g., and the
chloride of calcium gained 0*080 g. The total percentages were
10-12 per cent, loss, or 5*5molecles, and 9*23 per cent, gain, or
5*00 molecles of water.
Fourth Experiment. — 0*779 g. of dry cerebrinic acid at I'lS'^ lost
0*006 g,, and the chloride of calcium gained 0*0125 g. At 180°
the substance was completely melted, and lost 0*033 g., while the
chloride of calcium gained 0*037 g. At 210°, the current of air
being stopped as before until the tube had somewhat cooled down,
the loss was 0*0535 g., and the gain 0*030 g. The totals lead to
a loss of 11 67 per cent., or 6*347 molecles of water, and to a gain of
9*98 per cent., or 5*43 molecles. Oxidation may, as in the former
case, account for this discrepancy.
These caramels also were almost entirely soluble in ether.
Tabular Vieiv of the Data concerning the Caramels of Cerebrinic
Acid.
Dry
Amount.
Temp.
Per cent.
Loss.
Total
percent.
Loss.
Molecles.
Per
cent.
Gain.
Total
per cent.
Gain.
Molecles
of H2O.
1*613
210
7*55
7*55
4*10
6.38
6*38
3-47
1*221
160
2*46
2-70
200
5-32
7*77
4*227
4*60
7-30
3*97
1-073
150
1-12
1-77
200
2*66
1-86
200
6*34
10*12
5*504
5*60
9-23
5*02
* -779
145
•77
1*60
180
4*24
4*75
210
6-66
11*67
6*347
3*63
9*98
5-43
3. Sulphurised Principles, Sphero-cerebrin, and others.
Mode of sejjarating these Substances from the Cerebrin Mixture. —
The cerebrin mixture (that part of the white matter which
remains insoluble when it is exhausted with ether) is dissolved in
hot spirit, and to the solution a hot solution in spirit of acetate of
lead is added as long as a precipitate is j^roduced. Ultimately a
little ammonia is added, and the solution is filtered hot from the
precipitate. The latter contains the bodies here to be con-
sidered.
NITROGENISED NONPHOSPHORISED PRINCIPLES. 181
The crude Lead Compound^ to he hereafter referred to as 'Bark Lead
Salt/ Ox-Cerehrins. — This body was exhausted with boiling spirit,
the extraction being repeated an indefinite number of times ; all
that which spirit extracted will not be considered any further in
this place. The matter insoluble in spirit was now extracted
with benzol until nothing further dissolved. There remained in-
soluble a lead compound which had a peculiar ash-grey colour, re-
minding the observer at once of the probability that a small part
of the lead had by some means or other been converted into
sulphide.
- Quantation of Lead and Organic Matter and of Sulphur and Phos-
phorus in the Dark Lead Salt. — {a) 1*2992 g. decomposed with
hydrothion in hot spirit gave 0"3490 PbS, equal to 23-27 per
cent. Pb, and therefore about 76 '73 per cent, of organic matter.
{h) 1-4826 g. burnt with soda and mercuric oxide in a tube, etc.,
gave 0-1749 BaSO^, equal to 1*62 per cent. S, and 0*1574 pyro-
phosphate of magnesia, equal to 2-97 per cent. P.
Attempt to isolate the sulphurised Principle, the Presence of which
tvas demonstrated hj the foregoing Analysis. — The alcoholic solution
which had been obtained in analysis [a) of the foregoing para-
graph deposited much organic matter on cooling ; this was redis-
solved by heat, and in the hot solution a precipitate was i3roduced
by barita water. The precipitate was exhausted by boiling spirit,
and the extracts were disregarded. The matter insoluble in spirit
was dissolved in benzol, and the solution separated from the
insoluble portion. The benzol solution was concentrated by dis-
tillation, and precipitated by absolute alcohol. The precipitated
body was dried and analysed.
0-3068 (being almost the entire product of this first operation)
fused with nitre-flux, etc., gave 0-0288 BaSO^=l-32 per cent. S.
This experiment proves that the sulphurised principle contained
in the lead salt can be transformed into a barium salt soluble in
benzol. This salt contained less sulphur than the original lead
salt. When it is compared to other sulphurised barium compounds
of similar properties, which I shall have to describe below, it is
seen that in the former the sulphur amounts to a much lesser
percentage than in the latter. From a third series of observations
to be related, it will be seen that the brains of young animals
contain at least one sulphur body, which is so labile that on
standing in its ether solution it deposits sulphur in the metalloid
182
CHEMICAL CONSTITUTION OF THE BBAIN
state, and in crystals too, which are needles as well as octahedra.
AVe have therefore three distinct aper(;us, proving the presence of
sulphur compounds (other than albuminous ones) in the brain.
Decomposition of the Dark Lead Salt (Ox-Cerehrins) hij Oxalic Acid '
in boiling Spirit. — It was evident that for the study of the sulphur
compound in the lead salt the application of sulphuretted hydrogen,
although apparently quite successful in the above experiment,
would have to be avoided. A quantity of the salt was therefore
boiled with double that amount of crystallised oxalic acid, which
theory indicated as necessary for the transformation of all lead
into oxalate, in a flask fixed to a refluent cooler, until the salt ap-
peared white and all grey colour had disappeared. The solution
was now filtered from the precipitate.
InwluhU Lead Salt [Oxalate). — It was exhausted with spirit,
then washed with boiling water, suspended in water, and treated
-with dilute nitric acid. On heat being applied a reaction ensued,
the oxalate dissolved, and a small quantity of lead sulphate re-
mained insoluble. It weighed 0-0348 g., equal to 0-0037 g. S in
20 g. salt, or 0-0185 per cent. S in lead salt. This shows that of
-the 1'62 per cent. S contained in the lead salt, only a minute
proportion can be present as sulphuric acid, or in the shape of a
metallic sulphate. Possibly even the small quantity found was
derived from the oxidation of the black sulphide, which gives to
the salt its dark colour.
The Organic Matter from the Lead Salt (total mixture of at least
three bodies) was crystallised from the spirit containing the
excess of oxalic acid, washed, and pressed. It was next recrys-
tallised from spirit, and appeared white and voluminous. It was
analysed in order to ascertain whether it contained sulphur and
phosphorus, with the following result :
(a.) 0-8668 fused with flux and caustic soda gave 0'0340
BaSO^ = 0-54 per cent. S.
(h.) The solution filtered from the BaSO^ gave, further,
0-0638 pyrophosphate of Mg, equal to 2-06 per cent. P.
The organic mixture was now extracted with cold benzol, and
filtered by air-pressure through a tubular filter. The benzol ex-
tracted a small quantity of a body which was recrystallised from
spirit and crystallised in needles. The part insoluble in benzol
was also recrystallised from spirit. It was then recrystallised
from hot benzol, and deposited entirely on cooling. After having
NITROGENISED NONPHOSPHORISED PRINCIPLES. 183
been freed from benzol it was dissolved in about a litre of abso-
lute alcohol, and cooled to 38°. A body was deposited in heavy
sphero-crystals, and adhered to the glass, so that the solution could
be easily decanted clear from it. This was repeated, and all
crystals were collected. They represent the body which in the
following is termed splierocerehrin.
4. Spherocerebrin.
The name is intended to indicate that the body is a cerebrin
crystallising in sphero-crystals. It was twice crystallised frac-
tionally from absolute alcohol. The solution having been filtered
clear, was now allowed to cool, while the temperature was read
off by an immersed thermometer ; it began to form a deposit at
50°, of which the main bulk came down between 43° and 41°.
When the deposition had ceased, the spherocerebrin was
filtered off.
Seen under the microscope, it appears in round balls of very
uniform size, which all have a peculiar three-branched mark in
the round field. When the balls are rolled, it is seen that they
contain three wedge-shaped fans each ; the side of such a fan
shows radiating needles ; three such wedges are united with
their sharp straight edges at a line representing the diameter of
the ball. By gentle pressure the balls break almost regularly
into the three wedge-shaped segments.
Spherocerebrin with oil of vitriol, on standing, gives only a
very feeble reddish colour attached to flakes. It is free from
sulphur, and contains only unweighable traces of phosph(^rus
(when 0*4462 were analysed). On elementary analysis it gives
data which are collated in the following synopsis :
Percents, Atoms.
C 62-75 58
H 11-08 123
N 1-23 1
O 24-94 17-3
100-00
In its main features spherocerebrin resembles cerebrinic acid,
but it contains over this (theory C^gH^j^NOc,) an excess of 8H
and 80, or 8 hydroxyls. Spherocerebrin differs, therefore, from
cerebrinic acid not only by its crystalline shape, but also by its
percentic composition.
184 CHEMICAL CONSTITUTION OF THE BRAIN.
5. Principal and Second Product from Dark Lead Salt.
The term ' principal ' here refers to quantity ; 20 g. of lead salt
gave 15-49 of organic product.
The absolute alcohol solution had been filtered at 40°. At 37°
cumuli of clouds formed under the bulb of the thermometer. At
36° the entire liquid was turbid. At 35° all translucency was lost,
and the crystals projected from margin into fluid. At 34° the
surface became concave, and the marginal ring of deposit was out
of the fluid. The particles aggregated, and the fluid became again
translucent. At 32° clouds of deposit sank, and the temperature
remained stationary for some time. At this point the deposit
was separated by filtration.
At 30° to 29° a new cloudiness arose, and a deposit began to
adhere to the glass. This second deposit included almost all of
the matter which was in solution in the alcohol. These processes,
therefore, resulted in the separation of the organic matter present
in the lead com-pound in the greatest quantity, into two bodies,
one of which, tlie least soluble in alcohol, was recrystallised by
itself ; while the other, the most soluble in absolute alcohol, was also
recrystallised by itself.
We have, therefore, at this stage the organic matter in the lead
salt separated into the following five products, arranged in the
order of decreasing quantities :
(1) Principal product (not yet unitary) - - 9"59 g.
(2) Second product (more soluble than foregoing) 4*04 „
(3) Spherocerebrin (least soluble in alcohol) - 1*50,,
(4) Needle body soluble in benzol - - - 0-36 „
(5) Sulphurised compound - . _ . —
The Piinclpal Product from Dark Lead Salt (Ox-Cerebrlns). — Water
expelled at 90° = 3 -4800 percent. This body is a mixture of a
phosphorised principle with at least one cerebrin-like body. If we
assume all the phosphorus to be present in the shape of 1 molecle
of dineurostearyl-glyceryl-neuryl-phosphatide (C\^Hg3NP0g), and
deduct this molecle from the complex of molecles obtained as an
empirical formula with P = l, then we obtain a residue with more
than 7 atoms of nitrogen, which, divided by N = l {i.e., by 7'36),
gives a formula not unlike a cerebrin body with 3 nuclei of
neurostearic acid, but with a quantity of oxigen which is too
large to satisfy that hypothesis simply.
NITROGEN ISED NONPHOSPHOBISED PRINCIPLES. 185
Synopsis of the Percentages found and the Hypotheses applied.
Residue,
Percents.
^ At. Wgts.
■fP = l.
C,,H33NP08
deducted.
c
59-28
4-94
449
405
55-02
H
10-09
10-09
917
843
113-3
N
1-30
0092
8-36
7-36
1
P
0-37
0-011
1
0
0
0
28-96
1-81
164
156
21
100-00
We have, therefore, in the cerebrin residue of this mixture a
still larger number of atoms of oxigen than in spherocerebrin ;
and this fact may ultimately lead to an explanation of the add
character of these compounds, which enables them to form firm
compounds with bases, such as the cerebrosides of the phrenosin
type do apparently not form.
The second (more soluble in absolute alcohol) product, weighing
4-04 g. (see the list above given), has not yet been analysed. It
is not known whether it is or contains the sulphurised principle,
the existence of which in the mixture has been proved above.
This inquiry must be left to the future.
C. SUBGROUP OF CEREBROSULPHATIDES OR NON- ALBU-
MINOUS IMMEDIATE PRINCIPLES CONTAINING SULPHUR
AS AN ESSENTIAL INGREDIENT.
Introduction. — A sulphurised principle had already been dis-
covered in the lead salt from ox-cerebrins, regarding which some
preliminary information has been given in a previous chapter. I
may also here repeat the notice of an interesting observation
already alluded to on p. 182, nam3ly, the deposition of metalloid
crystallised sulphur from extracts of brains of young animals. I
have lately again expressly tested the phosphorised substances
which I have isolated and named, and found that they do not
contain sulphur as an essential constituent. A gramme of pure
kephalin gave only an unweighable trace of barium sulphate ; and
so with myelin and lecithin. The sulphur-compounds to be
described in the following were educed with the aid of barita.
Human mixed Cerehrlns. — Barita Process applied for their Separa-
ation.—l applied the barita process in this case in the hope that.
186 CHEMICAL CONSTITUTION OF THE BRAIN.
as others had by its means obtained cerebrin matter free from
phosphorised matter, I might be equally successful. My experi-
ment was, however, so far different from that of others, that I
carefully avoided the possibility of the barita acting chemolyti-
cally upon the cerebrin-substances. The barita was therefore
added only as long as it produced a precipitate, and never in
excess, so as to be in solution in any appreciable quantity. I
may at once state that in no single instance out of four experi-
ments were the cerebrins obtained free from phosphorus.
The Process. — 600 g. of human mixed cerebrin-substances were
dissolved, each 100 g. in 3 litres of hot alcohol. To the boiling
solution barita-water saturated at the ordinary temperature was
added in a thin stream, so as not to interrupt the boiling of the
alcohol. Each 100 g. had 450 cc. barita-water added. The
mixture was boiled for a few moments, and then the solution
decanted from the adhesive preciintate. The latter became
hard on cooling, and was detached and powdered. It was now
exhausted with boiling alcohol ; many extractions and pulverisa-
tions were required to withdraw all the matter which alcohol
would redissolve.
Nature of the Precipitate p'ocluced by Barita. — The barita-water
does not produce a precipitate in the hot alcoholic solution of the
cerebrin mixture by virtue of its water, as was specially proved
by a blank experiment with water only. It precipitates, in the
first place, in combination icith barita, a bod}^ (or mixture of bodies)
which is soluble in cold benzol, and which will be treated of
immediately. At the same time there fall down portions of the
several cerebrin bodies, phrenosin, kerasin, etc., of which another
portion remains in solution, and which are then again extracted
from the precipitate by boiling alcohol without retaining more
than traces of barita, precipitable by carbonic acid from the
alcoholic solution. These latter bodies are insoluble in cold
benzol, and separable thereby from the stearoconote or mixture
of barita compounds.
It must at once be pointed out that the matters soluble in cold
benzol cannot be separated from those insoluble in benzol by one
operation only. AVhen benzol has acted upon the powdered
particles of the precipitate, they seem to be covered by a layer of
matter insoluble in benzol, which prevents this solvent from
reaching the matters soluble in it, which are contained in the
JSITROGENISED NONPHOSPHORISED PRINCIPLES. 187
interior of the particles. It is therefore necessary to extract the
j)Owder alternately with cold benzol and hot alcohol, and suffi-
ciently often until all the matter is dissolved in hot alcohol and
cold benzol respectively, or remains insoluble in either.
Matters soluble in cold Benzol. — The benzol solution obtained in
the manner described in the foregoing is clarified by repose, filtra-
tion; and decantation, until on standing further it remains per-
fectly clear and brilliant. It is then concentrated to a small
bulk, with the precaution of keej^ing the solution perfect. It is
now treated with absolute alcohol as long as a precipitate or tur-
bidity is thereby produced. The precipitate is further extracted
by boiling absolute alcohol, and dried in vacuo over oil of vitriol.
The alcoholic mother-liquors, particularly those obtained by boil-
ing, contain a quantity of a body which appears in curved needles,
and therefore will be designated meanwhile as curved needle body.
The Earita-Compo^md soluble in cold Benzol and insoluble in
Alcohol. — It was dried over oil of vitriol, at 70° in air-bath before
analysis. It was a coloured powder, and on elementary analysis
gave data which are collated in the following synopsis :
Synopsis of Data :
Elements.
Percents.
H-by At. Wgts.
-4-by S = l
c
30-86
2-5716
20-57
H
4-88
4-88
39-
0-74
0-0528
0-42
S
4-00
0-125
1-
Ba
35-30
0-257
2-
P
2-55
0-082
0-65
0
21-67
1 :354
10-
100-00
The study of the relations of the atoms to each other gives at
once some interesting information. The sulphur is to barium as
1 : 2, a fact which became already apparent in the course of the
analysis. Carbon and hj^drogen are to each other as 1 : 2 very
nearly, indicating the presence in the compound of radicles of the
fatty series. Phosphorus and nitrogen stand apparently in no
relation to each other, and in none to the sulphur or barium. The
body is unquestionably a mixture, but may be of substances having
some analogy with each other. At least the bearing of the pro-
duct towards solvents would support such an hypothesis.
Severed smaller preparations obtained in a manner similar to the
188 CHEMICAL CONSTITUTION OF THE BRAIN
process described in the foregoing were analysed, and gave, one,
32-97 per cent. Ba, 1-06 per cent. S, and 1-15 per cent. P ; the
other, 22-14 per cent. Ba, and 1*95 per cent. S. They evidently
contained a lesser proportion of the sulphurised body than the
main preparation.
D. SUBGROUP OF AMIDOLIPOTIDES, OE NITROGENISED FATS,
1. Bregenin : ITS Isolation and Properties.
I have isolated the principle from both human and bovine
brains. It crystallises, fuses like a fat, and contains nitrogen. It
is extremely soluble in several reagents, and does not combine
with acids, alkalies, or salts.
Mode of Isolation. — Human cerebrins in hot alcoholic solution
were treated by barita-water. The precipitated cerebrin bodies
were freed from barita by sulphuric acid in alcohol and recrys-
tallised. The most soluble part contained the bregenin. From
this, sphingomyelin was removed by cadmium chloride ; the
resulting solution was now evaporated to dryness and extracted
with cold benzol ; kerasin remained undissolved, while bregenin
and some other matters dissolved in the benzol. The matters
dissolved in benzol were treated with boiling ether in a suitable
apparatus, when a body remained insoluble in boiling ether, while
two bodies dissolved ; one was deposited from the ether on cool-
ing— krinosin, to be described below — while another remained
dissolved in the cold ether, namely, bregenin. After removal of
the ether the bregenin was dissolved in a minimum of watery
spirit, filtered hot, and allowed to crystallise. When after re-
peated crystallisation, removal of the mother-liquor by pressure
between bibulous-paper, etc., the product consisted of white
microscopic leaflets and curved needles only, it was considered
pure and analysed. Its solution in spirit does not give precipi-
tates with cadmium or platinum chloride, or with lead acetate,
with or without ammonia. As long, therefore, as in a supposed
or reputed solution of bregenin such precipitates are produced,
they have to be removed as impurities. It is much more soluble
in absolute alcohol than in watery spirit ; from the former it is
deposited as a white solid mass, when the solution is concentrated ;
when dilute no deposit at all may take place ; it is useful to dilute
the hot alcoholic solution with boiling water until a permanent
NITROGENISED NONPHOSPHORISED PRINCIPLES. 189
turbidity is produced, and allow the mixture to cool. Good
crystals are obtained from such a solution.
From a concentrated spirit solution bregenin crystallises be-
tween 50° and 25°. At 30° it is not completely deposited. A
dilute spirit solution becomes hazy about 40°, and begins to
crystallise only at 25°. When thus deposited slowly it appears in
balls and irregular masses, without any curved needles. These
balls can be transformed into needles by resolution in spirit and
rapid cooling down of the solution.
Physical and Chemical Properties of Bregenin. — Besides the
characters already described in the foregoing, bregenin has the
following diagnostic properties. When heated in a water-oven it
becomes a little coloured, and then fuses below 98° to an oily
fluid. On cooling it solidifies to a hard mass, which is not plastic
like fat or wax, but splinters, when cut, in all directions, and is
highly electrical. Its fusing-point is between 62° and 65°. When
the fused and recongealed body in the filiform tube is again
heated, it becomes in part transparent about 62°, but a core
remains opaque until a higher temperature is reached, when all is
again fused. This is due to the viscosity of the fused body. At
the lowest fusing-point it is transparent, but so viscous as to be
hardly mobile, or only ver}^ slowly mobile ; with rising tempera-
ture it becomes as fluid as a molten fat, and the interval between
the point of fusion and the point of greatest liquidity is consider-
able. While it fuses and coalesces in the narrow tube at 62° to
65°, it flows down the sides of the tube only at 75° to 76°, and
then, on cooling, sets with a sudden aj)pearance of opacity at 58°.
These experiments were made with the purest specimen, which
had been kept in a fused state in the water-oven for hours. The
bearing of bregenin with water is very remarkable. When heated
with much water, it fuses like a fat, on the top of it. ' But on
agitation it increases in bulk and becomes viscogelatinous. This
hydration, which is completed only on long standing in the water,
causes it to increase in bulk considerably. When the swelled
mass, after decantation of all water, is heated on the water-bath,
it contracts, gives out water, and fuses as in its original state
while all water is being evaporated.
With oil of vitriol it gives no purple on standing ; the solution
remains a little yellow. When sugar-syrup is added to this
solution it becomes perfectly Avhite, and gives no vestige of
190 CHEMICAL CONSTITUTION OF THE BRAIN
purple. From this it is highly probable that bregenin does not
contain the radicles either of oleic acid or of sphingosin.
The fused bregenin, when quite cold again, can be powdered.
In this state it was subjected to elementary analysis, which
yielded the following data :
Synoj)sis of Analysis and Theory.
1. 2. 3. 4.
C 73-59 73-69 73-72 —
H 12-54: 12-92 12-56 —
N — — — 2-22
O — _ _ _
5. Mean. -^At. Wt. -^N = 1.
— 73-66 6-138 C^o
— 12-64 12-64 Hgi
2-15 2-18 0-155 N
— 12-52 0-781 O5
10000
This body, hregenin (from the Low German ' bregen,' head or
brain, the latter English word being probably a contraction merely
of bregen), C^oHg^NO^, is thus shown to be approximate in the
number of its carbon atoms to both the mononitrogenised phos-
phatides and to the cerebrosides. From the latter it is sharply
distinguished by the low amount of oxigen which it contains. It
has almost exactly the composition of the lowest phosphatide of
the lecithin group minus the phosphoric acid, for
C.„H,iNO, + PH30, = C,„H,3NP03 + H,0.
This might make us suspect that it was derived from such a
group by the mere loss of phosphoric acid, and consequently that
it might be a product and not an educt. But such a mode of
decomposition of a phosphatide has no analogy in the decomposi-
tions which have thus far been artificially produced. For in
these phosphorised bodies when they contained glycerol, this
alcohol remained mainly with the phosphoric acid. Supposing,
as a mere hypothesis, that bregenin did contain glycerol, then
there would be no room for neurin as the nitrogenised radicle.
In the absence of neurin, the nitrogenised radicle would have to
be one which does not occur in the mononitrogenised j^hospha-
tides, as far as they are known, and this would negative the
suggestion that bregenin might be derived from such a phospha-
tide by loss of phosphoric acid. Besides, such a hypothesis also
presupposes that the binding radicle is an alcohol like glycerol,
and that phosphoric acid is outside the nucleolar arrangement, and
merely attached as a side-chain — a theory which is clearly impos-
NITROGENISED NONPEOSPHORISED PRINCIPLES. 191
sible for those phosphatides, which, as I have shown, contain no
glycerol, and is therefore not probable for the others which con-
tain it.
Bregenin is obtained by the foregoing processes in small quanti-
ties only, and a great number of preparations were required to
yield material sufficiently pure for analysis. It is evident that
the greater part of the bregenin present in the brain must, in the
process of extraction, pass into the alcoholic and ethereal solution
containing lecithin and kephalin ; then it must at last remain
with the cholesterin, where we accordingly find it. And it is
separated from this only by treatment with caustic potash, which
chemolyses and removes the products of bregenin, while leaving
cholesterin unaltered. It will therefore be seen that much further
study will be required to elucidate the physiological quantities
and functions of this remarkable substance.
2. Krinosin, the Second Eepresentative of the New
Series of Nitrogenised Fats, or Amido-Lipotides : its
Isolation and Properties.
Mode of preparing Krinosin. — Dry, finely-powdered crude kerasin
as obtained from the process for the isolation of sphingomyelin
by cadmium chloride is exhausted in the ether extraction appa-
ratus by boiling ether. The solvent should be perfectly anhydrous,
and be renewed from time to time until it extracts nothing more.
For the extraction is but slowly completed, and in most cases
requires the apparatus to be kejDt in action for several days. The
hot ether solution deposits krinosin on cooling and standing as a
voluminous felted mass of long microscopic fibres, a wilderness of
mere lines, mostly without visible end. If the vessel has not
been agitated during the cooling process, the felted mass forms a
complete jelly with the ether. By strong agitation this structure
is destroyed, and there remain only a few lumps of felted matter,
looking like bluish paper pulp in an abundance of water. The
fibres, having been collected on a filter, dry to a hard mass, when
moisture, so easily collected by the cold j^roduced in the course of
the evaporation of the ether, is allowed to deposit upon them ;
when onl}^ little moisture has been in contact with them, they
dry to a white spongy matter, which is just a little waxy on com-
pression or when rubbed with the finger-nail. When the fibres,
taken out of the anhydrous ether, are dried in vacuo over oil of
192 CHEMICAL CONSTITUTION OF THE BRAIN.
vitriol, they become a perfectly white and pulverisable mass.
When this powder is now heated in a water-oven to 98° for some
time, it becomes somewhat plastic and assumes a yellowish colour.
After cooling it is again hard and perfectly pulverisable, but re-
tains the colour acquired by the heating. Krinosin is insoluble
in cold, easily soluble in boiling alcohol. It gives no purple reac-
tion with sulphuric acid alone, and none with sulphuric acid and
cane-sugar. It is consequently not a cerebroside, and does not
contain any oleo-cholide radicle.
Synopsis of the Means of Elementary Analyses and Theory of
Formula.
Percentages.
.-^At. Wgts.
-f-Na = l.
c
70-94
5-911
38-1
H
12-28
12-28
79-
N
2-17
0-155
1-
0
14-61
0-913
5-8
Consequently the nearest formula warranted by the quantations
is C3gH-gN05. Leaving out of consideration a slight excess of
hydrogen, probably due to the substance not having been dried
at a higher temperature, but only in vacuo, there seems good
reason to suppose that krinosin is a homologue, in an isomeric
series, of bregenin, for, we have
Bregenin - C^oHgiNO.
Krinosin - C,sH..N05.
The homology is probably not in the same series, as the higher
carbonised bregenin fuses below 70°, while krinosin does not fuse
below the heat of boiling water.
E. SUBGROUP OF ALKALOIDS.
1. Alkaloids from the Human Brain.
1. Hypoxanthin. — The deposit from the concentrated solution
of the extractive matters of the brain soluble in water was ex-
hausted with HCl ; the solution was treated with excess of silver
nitrate, and the precipitate extracted to exhaustion Avith boiling
dilute nitric acid. The deposited hypoxanthin silver nitrate was
isolated and added to the preparation obtained by the following
process.
NITROGENISED NONPHOSPHORISED PRINCIPLES. 193
The extract was diluted and precipitated with mercuric acetate.
The precipitate was washed by repeated levigation in water, and
decomposed by H^S. The HgS was repeatedly extracted with
boiling water. The solutions were evaporated to a thick dark
syrup, and filtered. A first crop of impure hypoxanthin remained
on the filter. Addition of ammonia produced a second one.
Both portions were united, dissolved in dilute HCl, boiled with
animal charcoal, and reprecipitated by ammonia ; evaporation to
dryness and extraction of the ammonium chloride with water left
the hypoxanthin, which was now also transformed into silver
nitrate salt.
The united silver nitrate salts were treated with ammonia
during several days, and washed with ammonia-water on the
filter. The solutions contained little besides amnionic nitrate.
The last extracts contained a very small quantity of a body
soluble in ammonia, precipitated in flakes on evaporation, and re
dissolved in nitric acid on boiling, less readily precipitated on
cooling than hypoxanthin salts, and therefore more like xanthin
or guanin.
The silver hypoxanthin was decomposed by H^S, but the solution
remained black and unfilterable. It had to be boiled with animal
charcoal, and then became almost colourless. But the charcoal
retained much hypoxanthin, and had to be boiled with many new
portions of water before the whole of the base which could be
extracted was obtained. Some was no doubt lost in the charcoal.
The solution was acid and contained some phosphate from the
charcoal. It was treated with a little ammonia, filtered, evaporated
to dryness, and extracted with "cold water. The pure white crystal-
lised hypoxanthin was collected on a filter^ washed with cold water,
and dried. It formed crystalline white masses and granules.
2. Second Alkaloid. — The mother-liquor of the hypoxanthin,
treated with barita to expel the ammonia, and then with carbonic
acid, retained a large amount of barita in solution. The solution
gave precipitates with ferric chloride on boiling, and with phos-
phomolybdic acid. The barita was removed by sulphuric acid,
and the acid solution precipitated by phosphomolybdic. The
precipitate was decomposed with barita ; the filtrate was refiltered
three times during concentration.
The solution was acidified with HCl, and precipitated by AuClg.
The auric chloride salt was washed with water (the salt was very
13
194 CHEMICAL CONSTITUTION OF THE BRAIN
soluble in excess of HCl), and put in a vacuum to dry. On
analysis it gave the data contained in the following synopsis :
Synopsis and Computation of Analyses.
Percentages .
-7- by At. Wgts.
-V- Au = l.
N = l.
c
19-447
1-620
6-98
2-38
H
2-221
2-221
9-57
3-27
N
9-499
0-678
2-92
1-00
0
7-890
0-493
2-12
0-72
Au
45-743
0-232
1-00
0-34
CI
15-200
0-428
1-84
0-63
It is at once evident that the compound contains more gold than
could be present in the form of terchloride; for the 15-200 per cent.
CI require 28 073 Au ; the excess of Au, therefore, amounts to
17 '670. This may be supposed to have been present in the re-
duced metallic state. The salt is, therefore, one of those unstable
compounds which decompose during isolation and drying. Never-
theless, the consideration of the composition of the residue will
afford some, the only, means of judging of the composition of the
original alkaloid. From the computation of the figures obtained
by N = 1, and deducting the excess of gold, and referring the
possible organic molecle to a molecle of AuClg (which requires
the multiplication of the quotients by N = 1 by 5), we get
CijHjgNgOg.^AuClg. But calculating the salt which remains
after deducting the excess of gold as a hydrochlorate and auro-
chloride, containing 4C1 to lAu, we get —
Percentages.
^ At Wgts.
-f Au^l.
c
19-447
1-620
15-14
H
2-221
2-221
20-75
N .
9-499
0-678
6-33
CI )
15-200
0-428
4-00
Au/
21-055
0-107
1-00
Au
24-688
0
7-890
0-493
4-60
leading to a formula consonant to the general chemical theory of
gold double salts of Ci5H,oN^O„HCl,AuCl3.
3. Third Alkaloid. — This was left in the mother-liquor after
removal of the two previous bodies, and precipitated by phospho-
molybdic acid from the acidified liquid. The treatment of the
precipitate by barita yielded the alkaloid in solution, retaining
barita not precipitable by carbonic acid. The solution was pre-
cipitated by absolute alcohol. It remained a syrupy coloured
NITROGENI'SED NONPHOSPHORISED PRINCIPLES. 195
liquid, and did not crystallise after months of standing. It had
a marked smell of human sperma. It was so small in quantity
that no further research could be instituted upon it. After it
had been removed from the mother-liquor representing the ex-
tracts obtained by the mercuric acetate precipitation, there
seemed to be no further substance of an alkaloidal nature con-
tained in them.
2. Alkaloids contained in Ox Brain.
The presence of the alkaloids in the water-extracts of brain-
matter is indicated by the precipitates which they give with
AuClg, I in KI, HgCl2 in KI, picric, tannic, and phosphomolybdic
acids.
They may be obtained as shown in the preceding pages by the
application of the phosphomolybdic acid process, HgCU process,
and collaterally by the basic lead acetate process (for inosite).
The latter process, however, does not yield them if preceded by
the phosphomolybdic acid process. They may be separated when
in the free state by evaporation to a syrupy consistency when the
hypoxanthin is deposited while the second base is uncrystal-
lisable. The same remarks apply to a mixture of the hydro-
chlorates, the hypoxanthin compound in this case being obtained
in a crystalline form. Neither the watery solution of the free
hypoxanthin nor that of its hydrochlorate gives a PtCl^ precipitate.
The hypoxanthin may be purified by dissolving in boiling water
and addition of excess of silver nitrate and nitric acid. The
precipitate so formed gradually dissolves in hot nitric acid, and
from the filtered solution deposits on cooling in a mass of homo-
geneous needles. Obtained in this way it is pure, as testified by
the following analysis :
. . Analyses and Theory.
Percentages.
-fAt. Wgts.
-Ag=l.
C = 19-746
1-645
5-2
H = 1-539
1-539
4-8
N = 23-516
1-679
5-3
0 = 21-099
1-318
4-1
Ag = 34-100
0-315
1-0
^C-H^N.O-f AgNOg.
A combination of the hypoxanthin with silver was also obtained
by precipitation of its solution with ammoniacal silver nitrate ;
13—2
196 CHEMICAL CONSTITUTION OF THE BRAIN
it contained 6046 per cent, silver, whereas had it been pure
C-H^N^OAg^„ it should have contained 61*7 per cent.
The hydrochlorate of the second base is soluble in alcohol but
precipitated by ether ; on the whole it is a perishable compound.
In one experiment Avhere the hypoxanthin had been removed
from a mixture of the two bodies, this second base was again
passed through the phosphomolybdic process ; the product gave a
precipitate with AgXO^ in the presence of HNO^, which proved
to be a further quantity of hypoxanthin salt. Thus purified
the second base was again passed through the j^hosphomolybdic
acid process, then converted into gold chloride salt and analysed ;
it yielded —
C 27-37
H 3-71
N 11-88
. ' O 13-836
Au 31-864
CI 11-34
100-000
Computation of Analyses. — In this compound the gold stands to
the chlorine as 1 : 2 ; deducting now the gold and chlorine and
recalculating the percentages, we have :
Percentages. —-At. Wgts. -^N=1.
C = 48-18 4-015 2-7
H = 6-53 6-530 4 3
N = 20-92 1-494 1-0
O = 24-37 1-523 1-0
= C3H4NO.
F. SUBGROUP OF AMIDO-ACIDS AND IMIDES.
1. Leucin and Allied Bodies ; Tyrosin.
The filtrates from the mercuric precipitates were freed from
mercury by hydrothion, and evaporated to a syrupy consistence ;
the S3TUP was treated with absolute alcohol, and thereby separated
into a portion soluble, and another insoluble, in alcohol.
Portion Soluble in Alcohol. — The alcohol was evaporated, the
residue treated with neutral plumbic acetate. The precipitate
consisted mainly of j^hosphate and chloi'ide, and was removed.
After this, basic plumbic acetate was added to the filtrate, and
NITROGENISED NONPHOSPHOKISED PRINCIPLES. 197
the precipitate collected and decomposed with hydrothion ; the
concentrated filtrate from the plumbic sulphide on treatment with
absolute alcohol gave a crystallisation of inosite.
The mother-liquor of this inosite, freed from alcohol by evapo-
ration and acidified, gave a small precipitate with phosphomolybdic
acid, thus proving that the mercuric acetate had not removed all
the alkaloid, but left some, which was precipitated by basic lead
acetate {third alkaloid).
The lead was now removed from the mother-liquor by an excess
of sulphuric acid, and the acid liquid extracted with large quan-
tities of ether at intervals. The ethereal extracts, after removal
of the ether by distillation, left a mixture of acids ; of these,
acetic acid, introduced with the mercury and lead salts, was
evaporated on the water-bath. The remaining syrup consisted
mainly of lactic acid, but also contained some succinic acid, as will
be described more fully lower down.
Portion Insoluble in Alcohol. — This, on solution in a minimum
of water, retained a quantity of solid matter in suspension, which
was separated by filtration. The residue on the filter was pressed,
and on treatment with cold water gave to this solvent a matter
recognised as leucin and allied bodies. The matter insoluble in cold,
but easily soluble in hot water, was tyrosin.
The leucin was purified by precipitating the coloured impurity
out of its watery solution by means of mercuric nitrate, removing
the excess of mercury by hydrothion, evaporating the solution,
precipitating the acid liquid by ammonia, collecting and pressing
the precipitate in bibulous paper,- and recrystallising it from
spirit. The first crystals were pure leucin ; the second crystals
were, however, a different body, more soluble than leucin, and not
easily separated from leucin, on account of the similarity of pro-
perties. The last mother-liquor dried up, leaving a trace of matter.
The tyrosin was purified by hydrochloric acid and charcoal,
and precipitation with ammonia.
The mercuric nitrate precipitate from leucin was also decom-
posed, and seemed to contain onl}^ a small amount of alkaloid
No. 2.
Note on a Pecidiar Potassium Salt.
The liquid part of the portion insoluble in alcohol was diluted
with water, and treated with neutral lead acetate ; the precipitate,
198 CHEMICAL CONSTITUTION OF THE BRAIN.
decomposed by hydrothion, gave a syrup which, treated with
absolute alcohol, deposited a peculiar viscous colourless potassium
salt ; this was isolated, and, on account of its peculiar nature, the
presence of the potassium was specially proved by combustion
and platinic chloride. The part of the syrup soluble in alcohol
was not further examined. The filtrate from the neutral lead
acetate precipitate was treated with basic lead acetate, and the
new precipitate decomposed with hydrothion ; the concentrated
product was treated with absolute alcohol, and gave white inosite,
partly anhydrous, partly hydrated. In the mother-liquor of this
inosite nothing, apparently, but some alkaloid No. 2 remained,
The mother-liquors from which the foregoing bodies had been ex-
tracted and precipitated were freed from the impurities introduced
as reagents as far as possible and evaporated, and then formed a
nearly colourless, viscid, uncrystallisable mass ; this was distilled
in superheated steam, but yielded no glycerol. It gave a precipi-
tate with phosphomolybdic acid, from which a syrupy alkaloid,
smelling hke sperma, was obtained (alkaloid No. 3).
GEOUP OF IMMEDIATE PRINCIPLES, COMPOSED
OF THEEE ELEMENTS ONLY : ALCOHOLS, CARBO-
HYDRATES, AND ACIDS.
A. SUBaROUP OF ALCOHOLS.
Cholesterin.
Cholesterin is present in the brain in very large quantity. In
the process for the separation of the brain principles above de-
scribed it passes mainly into the solution containing the kephalins ;
these bodies have to be precipitated by lead ; myelin the same ;
the other phosphatides have then to be removed by cadmium
chloride. At last a mixture of cholesterin, with several other
bodies, amongst them bregenin, is obtained ; this mixture must
now be boiled with caustic potash in alcohol, to decompose the
admixtures, and retain their decomposition products in solution
while cholesterin crystallises. It is pressed and recrystallised as
often as necessary to give it its brilHant appearance, and its
melting-point, 145°. It is then the same body as that obtained
from human gallstones. It crystallises from spirit as monohydrate,
C26H44O + H2O, and loses the water at 100° or in vacuo. While
from spirit it crystallises in rhombic plates, it is deposited from
chloroform or benzol in anhydrous needles. It rotates polarised
light to the left. Ether solution at 15°: [a]D = -3M2° ; in
chloroform solution: [a]D = —36-61°. It is insoluble in water,
little soluble in cold watery spirit, easily soluble in from 5 to 9
parts of boiling alcohol, the more the stronger the alcohol is. It
can be distilled in a vacuum unchanged at a temperature of 360^
On distillation by heat under ordinary air-pressure it is partly
transformed into hydrocarbons. It combines with organic acids
200 CHEMICAL CONSTITUTION OF THE BRAIN.
when heated with them under pressure. When oxydised by
])ermanganate in acetic acid sohition it yields cholestenic acid,
^25-^40^4' similar acids with 5 or 6 atoms of oxigen. AVith
nitric acid it yields cholesteric acid, Cj._,Hj^O^, which is remark-
able, as it is also formed by nitric acid from cholic acid, and thus
it establishes a relationship between cholesterin and biliary acids.
With bromine cholesterin gives a product of addition, CggH^OjBrg ;
with concentrated sulphuric or phosphoric acid it yields a number
of hydrocarbons, cholesterylens, which are isomeric with each
other.
Cholesterin gives some very characteristic reactions. Treated
with concentrated sulphuric acid and a little iodine, it becomes
violet, blue, green, and red in succession. This reaction is useful
for recognising cholesterin under the microscope. When a little
cholesterin is evaporated with a drop of nitric acid at a gentle
heat, a yellow spot remains, which, if covered while warm with a
drop of ammonia, becomes red ; this red is not altered by the
addition of fixed alkali.
Reaction of Cholesterin with Oil of Vitriol and Chloroform ; Spectral
Phenomena of the Product.
The cholesterin was obtained from the brain, and fused at
147° C. A portion was dissolved in chloroform, and an equal
bulk of oil of vitriol added ; this produced a dark red coloration
both in the chloroform and in the acid.
The chloroform solution thus obtained presented the following
spectra :
The most concentrated solution obscured all but the red.
After it had been a little more diluted one broad band appeared
in yellow and green, and another in green to blue, but it had only
about half the intensity of the other.
AVhen it was still more diluted the broad band split into two
bands, the band in green to blue remaining.
The solution when poured into a dish became rapidly blue,
green, and at last colourless.
On evaporation to dryness and re-addition of sulphuric acid to
the residue, a sHght restoration of a dirty red colour took place,
but with chloroform no restoration ; the permanent colour of the
residue was a light green.
Reaction of Cholesterin ivith (Til of Vitriol and Glacial Acetic Acid.
The dark red-brown oil of vitriol solution of cholesterin when
thrown into glacial acetic acid dissolves entirely to a dark red
solution almost impenetrable to light. This solution presents
the following spectra :
Through the concentrated liquid only red passes ; when it is
more diluted two bands appear, one feeble, in red, the other
strong, in orange and yellow.
In the still more diluted liquid three bands appear, the solution
being brilliant ; the third band is broad, and reaches to F.
Hence the middle band of the chloroform solution was absent
from this ; the broad band of the chloroform solution was there,
but much stronger and broader. There was a powerful green
fluorescence. As the third band of the acetic acid solution is new
and does not occur in the chloroform solution, at least two coloured
bodies are produced in this reaction.
By oxidation with chromic acid in acetic acid, cholesterin yields
an acid of the formula 0^4113^^05, and other products.
Phytosterin, the cholesterin of plants, is the second isomer,
C2gH440 + H20; its fusing-point is 132° to 133^; its chloroform
solution turns to the left =[a]D = —34-2°.
Isocholesterin, OggH^^O is the third isomer, and occurs in the wool-
fat from sheep, together with the ordinary cholesterin. When
crystallising from alcohol it forms gelatinous masses ; from ether
it crystallises in needles. It does not give the reaction with oil
of vitriol and chloroform which cholesterin gives. It fuses at 137°
to 138°.
Paracholesterin, O^gH^^O + HoO, is the fourth isomer, and is
obtained from the fungus sethalium flavum. Fuses at 134° to
134 '5°. It differs from phytosterin only by its rotation being
less, namely [a]D= — 28'88°.
The quantation of cholesterin in the brain is connected with
many difficulties. It is not impossible that the bearing of cho-
lesterin with benzoic acid under pressure at high temperature,
200°, may be utilised for this purpose. The two bodies combine
and form an ether, which is almost insoluble in boiling spirit ; but
crystallises from ether in peculiar rectangular plates. The benzoate
of isocholesterin crystallises in needles. When such plates and
needles are obtained mixed, they can be separated by levigation
202 CHEMICAL CONSTITUTION OF TEE BRAIN
with spirit. This process therefore offers a means of separating
cholesterin from isocholesterin, when they occur mixed with each
other, as they do in wool-fat. It may possibly serve in some cases
of brain analysis, and for this purpose the reaction should be
borne in mind.
B. SUBGROUP OF CARBOHYDRATES.
Inosite.
Inosite occurs in the parenchyma of most tissues of the animal
body, but in largest quantity in muscle and the brain. It also
occurs in plants, e.g. in beans, and in Sauterne wine. It crystal-
lises as a dihydrate, CgHj^Og -f- 2H2O, and does not rotate the
ray of polarised light. It does not undergo the alcoholic, but
easily the lactic fermentation, and the lactic acid which results
is optically inactive. With nitric acid it yields a trinitrited,
and a hexanitrited substitution compound, CgHg(N02)30g, and
CgHg(NOo)gO(3. Inosite is completely precipitated from its solu-
tion by basic lead acetate ; the lead compound is decomposed by
hydro thion, and the solution evaporated to a small bulk. AVhen
toHhis alcohol is added, crystals of inosite are formed on standing.
The following is a good test for inosite. Evaporate the liquid to
be tested for inosite in a porcelain dish to the bulk of a few drops,
and then add a small drop of mercuric nitrate. This produces a
yellowish precipitate. When this is spread as far as possible over
the surface of the porcelain, and the dish is further heated with
great caution, there remains, as soon as all fluid is evaporated, and
provided that no excess of reagent has been added, a residue
w^hich is whitish-yellow at first, but soon becomes more or less
dark red, according to the quantity of inosite present. The
colour disappears when the dish gets cold, but reappears on re-
heating it gently. If, when the colour has appeared, the dish is
overheated in the slightest degree, the mixture undergoes a sudden
decomposition, though without incandescence, and becomes black.
Inosite is not capable of reducing Fehling's solution. Its bearing
with copper salts was little understood before the following obser-
vations on the subject were made.
Compound of Cerebral Inosite with Cupic Oxide. — When to a hot
solution of inosite (from ox-brain) a saturated solution of copper
acetate is added, a light green precipitate immediately ensues.
ALCOHOLS, CARBOHYDRATES, AND ACTDS 203
When copper acetate is added in excess, so that the filtrate has a
blue colour, and the mixture is warmed, almost all inosite is pre-
cipitated out of the solution. The green precipitate of inosite
copper can be heated with pure water, without more than traces
of copper dissolving in the water. The solution is colourless, but
gives a brown coloration with potassium ferrocyanide and acetic
acid. The light green precipitate of inosite copper {first precij)i-
tate) on being dried in the air-bath at 110°, became dark green,
nearly black, and was then analysed. It contained 47*11 per
cent, of Cu.
A compound of one molecle of inosite with three molecles of
cupric oxide, CgH^^^^d + 3CuO, of which the atomic weight would
be 182 + 238-2 = 420-2, requires 45-2 per cent.
The compound when hoUed with water is decomposed at the place
where the vessel, whether platinum or glass, is hottest. It is not de-
composed when warmed gently on the water-bath . When a solution
of inosite is evaimxited with excess of coj)per acetate on the water-
bath, all inosite becomes insoluble in the shape of the compound
described in the foregoing. The excess of acetate may be washed
out with warm water, but the precipitate may not be boiled in
platinum or glass over the free flame, as it forms a reddish-brown
adherent decomposition product. The compound is soluble in
acetic acid, with slight coloration, without residue. It is soluble
in ammonia, with a deep blue colour. A trace of matter remains
insoluble (which is not the case with the acetic acid) and may
explain the slight excess of copper found in the analysis.
When this compound was heated on platinum, it showed the
following remarkable bearing : it scintillated and deflagrated,
while evolving acid fumes. A red residue was left, which on
being thrown into the air took fire and burnt (pyrophorus) . Pro-
bably the three molecles of cupric oxide gave up half their oxigen,
and remained as cuprous oxide mixed with some carbonaceous
matter.
Second Precipitate. — On addition of more copper acetate to the
solution from which the first precipitate had been filtered, and
application of a gentle heat, a second precipitate ensued, which
was and remained green. Dried at 110° it gave on analysis, mean
of three quantations, 44-59 per cent. Cu.
When this compound in the state of powder was heated at the
margin, it took fire, and then burned spontaneously through,
204 CHEMICAL CONSTITUTION OF THE BRAIN
leaving a red residue. This residue also was pyrophoroiis when
thrown into the air.
Third Precipitate. — The mother-liquor of the second precipitate
was concentrated by evaporation, and formed a third precipitate.
This, after isolation, was dissolved in dilute ammonia, and repre-
cipitated by gentle eva2)oration. It was light green, and when
gently dried was analysed, and found to contain 40" 10 per cent.
Cu. "
A trihydrate of the cupric inosite requires 40-10 per cent, of Cu.
That this compound, CgHj2^6 + ^^^^ + 2^^20 (atom, weight
= 474-2) had really been obtained, was further proved by the
loss which it suffered on being dried at 110°, being equal to three
molecles of water. For the dry compound gave on analysis
45-38 per cent. Cu, while theory requires 45-2 per cent. Cu.
Fourth Precipitate. — This was obtained after the third, by the
same process as the latter. It was bright green, and dried at 100°
gave on analysis 45-68 per cent. Cu.
The inosite from ox-brain used in the foregoing research was a
finely crystallised, on the whole very pure specimen. Nevertheless,
the first precipitate was somewhat impure, as indicated by its
2)hysical properties and its composition. But the precipitates
Nos. 3, 4, were so pure that their analyses yielded almost theo-
retical results.
It was therefore very surprising that an equally well crystallised
and apparently perfectly pure specimen of inosite from human brain,
on being mixed and treated with copper acetate like the inosite
from ox-brain, should yield totally different and greatly varying
results.
The first p}recipitate, dried in vacuo over sulphuric acid, lost
at 110° 1-53 per cent. H^O, and contained 47*48 per cent. Cu.
The second precipitate, dried in vacuo, etc., lost at 110° 1*77 per
cent. H.,0, and contained 51-23 per cent. Cu.
The mixture, on further evaportion, formed a third, and after
its removal only an insignificant fourth precipitate, although after
addition of copper solution it formed a fifth and a sixth precipi-
tate, which contained more copper than the first precipitate.
The third precipitate, dried in vacuo over oil of vitriol, lost at
110° 2*85 per cent. H._,0, and contained 47*73 per cent. Cu.
The fifth and sixth ptrecipitates, dried in vacuo over sulphuric
acid, lost at 110° 1*34 per cent. H^, and contained 49*81 per
cent. Cu.
ALCOHOLS, CARBOHYDRATES, AND ACIDS. 205
These precipitates, therefore, contained about two, or four, or
six per cent, more copper than corresponds to tricupric inosite.
When they were dissolved in ammonia and the solution evapo-
rated cautiously, discoloured products were obtained. From
these data I conclude that the inosite from the human brain is
either altogether different from that contained in the brain of the
ox, or is accompanied by another similar carbohydrate of less
stable quality. In any case, the subject calls for further investi-
gation.
Inosite is a hexadynamic alcohol, and forms, as we have seen
above, two nitrite ethers, a hexanitrite and a trinitrite. Similarly,
though probably not by substitution of hydrogen, it forms a
tricupric compound ; and perhaps at the same time a small quan-
tity of a hexacupric one, if the compounds with more than 45*2
per cent. Cu have not to be considered as combinations with
bodies other than inosite.
C. SUBGROUP OF (NONNITROGENISED) ORGANIC ACIDS.
1. Lactic Acid.
The mixed acids were heated on the water-bath until acetic
acid was expelled, redis solved in water, and neutralised while hot
with freshly prepared zinc carbonate. The zinc salt was crystallised
and recrystallised an indefinite number of times, until perfectly
white, crystallised throughout, and homogeneous. During these
operations a coloured matter became insoluble, and had to be
removed by repeated filtration. The crystallised salt was found
to be pure zinc lactate, containing the variety of lactic acid known
as lactic acid from flesh, or sarkolactic acid. Not only did the salts
yield the particular amount of water of crystallisation which dis-
tinguishes them from the zinc salts of the fermentation lactic
acid, but the free acid itself showed the power of polarising light,
which is not possessed by the product of the fermentation of
milk.
Summanj of Analyses of Zinc Lactate from Human Brain.
The salt was dried in vacuo until it lost no longer in weight,
and then at 110° until constant. Two specimens showed a loss of
water of crystallisation amounting to 12-82 per cent., and 12*90
per cent. This corresponds to the theory of 12*82 per cent.
water of crystallisation.
206 CHEMICAL CONSTITUTION OF THE BRAIN
In the same hydrated salt, dried in vacuo, the zinc was estimated
by precipitation with carbonate in the usual manner, and found
in two experiments to amount to 23-24 per cent,, and 23 "28 per
cent. ; while theory requires 23-35 per cent. Zn.
Summanj of Analyses of Zinc Lactate from Ox Brain.
. The salt, after having been dried in vacuo over oil of vitriol,
lost 12-80 per cent. HoO at 110°.
The zinc was estimated in the anhydrous salt by precipitation
and ignition, and found in two experiments to amount to 26*69
j)er cent., and 26-69 per cent. Theory requires 26-79 per cent.
Zn in the anhydrous salt, CgHj^O^Zn. The formula of the
hydrated salt is Zn{C^B.Jd.^.^{B.fi).^,
Physical Peculiarities of the Lactic Acid from Brain and its Zinc Salt.
When the lactic acid as obtained from the ether extract, a state
in which it was yet yellowish and gave out an odour, was de-
colorised by animal charcoal, and a somewhat concentrated
solution of it was placed in a tube, of 220 mm. in length, and
containing about 26 cc. of fluid, and subjected to the influence of
the polarised ray of yellow light in a Wild's polaristrobometer, it
was found to turn the plane of polarisation to the left (to the
measured extent, in the particular instance of an acid of uncertain
strength, of V 20').
The acid was next transformed into zinc salt by boiling with
zinc oxide, and the solution of salt was evaporated to the same
volume as that occupied by the free acid. It now turned the
plane of polarisation still to the left, but to the extent of 3° 15'.
Thus the rotation from 0 to the left had been much increased,
more than doubled, by the introduction of the zinc and the
attendant thermal and hydric operations.
The zinc salt, which had been obtained in a state of purity, as
proved in the previous j^aragraph, was dissolved in water and
decomposed with hydrothion ; the free acid was concentrated and
became a colourless syrupy liquid ; in this state it was not per-
fectly brilliant, but had a slight haze, probably from a trace of
finely divided sulphur. It was therefore allowed to stand for
two months in a quiet place, and when the trace of particles had
completely deposited, the clear part was isolated by decanta-
tion.
ALCOHOLS, CARBOHYDRATES, AND ACIDS. 207
This acid, on being placed in a tube of 100 mm. in length into
the polaristrobometer, now turned the plane of polarisation to the
right, in the particular instance of an acid of uncertain concentra-
tion, to the extent of 2° 17' (average of seven observations).
I have made no attempt to determine the specific rotating-power
of the lactic-acid from the brain. The reason for this is the circum-
stance first observed by Wislicenus, that the rotating-power of sar-
kolactic acid changes under a great number of influences, such as
heat, water, and time. Thus free sarkolactic acid, when dried over
sulphuric acid in vacuo during 21 months, is transformed into a
mixture of lactic acid, CgHgO^ (16-50 per cent.); anhydride,
C^HioOg (84-19 per cent.) ; and lactide, CgH^O^ (16-04 per cent).
The solution of this mixture turns the plane of polarised light to
the left (ft)= -85*93°. It is probable that all three products
on treatment with water are transformed back into sarkolactic
acid.
The watery solution of sarkolactic acid as ordinarily obtained,
directly after extraction and concentration, shows a considerable
polarisation to the left. This power is suddenly and greatly
diminished after every addition of water or spirit, but on stand-
ing it rises again, without, however, reaching its former value.
The diminution of specific rotatory power by dilution is the
greater, the more concentrated was the solution used for dilution,
that is to say, the greater was the dilution in proportion to the
strength of the original solution. These changes are due to the
presence of anhydrides and lactide. And as every preparation of
sarkolactic acid contains these anhydrides, according to Wisli-
cenus, pure sarkolactic acid, as a preparation, does not exist, and
therefore its specific rotatory power, which this author surmised
to be to the right, cannot be accurately determined.
I have shown above that pure sarkolactic acid prepared from
the zinc salt turns the plane of polarised light freely to the right.
But the solution of the zinc salt from which this acid was pro-
duced turned energetically to the left. The saturated normal
solution of zinc salt turns the plane of polarised light steadily
7° 7' to the left. In over-saturated solutions the turning faculty
is not increased, as might be supposed, but, on the contrary, is
diminished.
The polarising faculties of sarkolactic acid, its hydrate, zinc
salt, and anhydride may be described as follows :
208 CHEMICAL CONSTITUTION OF THE BRAIN
Turning farthest to the right - C3Hg03.
Turning less far to the right - CgHgOg + H2O.
Turning least to the left - - 2(C3H503)Zn.
Turning more to the /^/Z - - 2(C3H5()3)Zn + 2H,0.
Turning farthest to the left - CgHjoO^.
Peculiarities of the Calcium Salt of Lactic Acid from Hmian
Brain.
This salt was made from pure lactic acid obtained as above de-
scribed. Its solubility in water was so great that attempts at its
crystallisation from this solvent were foiled by the solution setting
to a solid mass. It was consequently recrystallised from strong
spirit. When dried in air, it was a light voluminous spongy mass
of crystals, which on being dried at 105° lost 21-2 per cent, of
water of crystallisation, and contained 14-14 per cent, of Ca.
These data do not correspond to any of the recorded data con-
cerning this salt, which recorded data themselves differ from each
other, or, on the assumption that there was only one hydrate,
contradict each other. It is noAv assumed by some that the sarko-
lactate of calcium, as commonly obtained, has the formula —
, 2Ca,4(C3H^03) + 9H2O - - water = 27 '09 per cent.
Formerly, however, a hydrate was mostly
described as containing water - - = 24*83 per cent.
The hydrate just described contains water 21*2 percent.
Now, the second salt corresponds to one with 4 molecles of
water of crystallisation, while the last leads to no even proportion
between salt and water of crystallisation, but is intermediate be-
tween the salt containing 4 molecles and a hypothetical salt
containing 3 molecles of water, which requires 19-89 per cent.
The new salt must therefore be considered either as a compound
or mixture in nearly molecular pro2)ortion of the salt, containing
24-83 per cent., with the hypothetical salt containing 19*89 per
cent, of water, or, in more simple terms, as a salt consisting of
2 molecles of anhydrous lactate and 7 molecles of water of crys-
tallisation, requiring 22*35 per cent. H^O ; possibl}^ a lower
homologue of the salt with 9 molecles of water. The dej^ression
of the water by mere admixture of anhydride was improbable,
owing to the uniform character of the crystallisation.
I am, therefore, of opinion that there are at least three, if not
four, different crystallised hydrates of calcic lactate, and that the
ALCOHOLS, CARBOHYDEATES, AND ACLDS. 209
amount of hydration is probably dependent upon the concentra-
tion of the sohition if it be a watery one, or upon the aquosity of
the solvent if it be spirit.
The preparations of lactic acid from the brain of man and the
ox, which I have described above, leave no room for doubt regard-
ing their nature ; they are specimens of the one optically active
sarkolactic acid, yielding the precisely characteristic zinc salt ;
that they did not yield the ordinary calcium salt is of little con-
sequence, as the question of the composition of the calcium salts
of sarkolactic acid is not exhaustively answered. Whatever may
be the issue of the discussion regarding the constitution of the
different lactic acids, the facts now ascertained regarding cerebral
lactic acid cannot thereby be affected.
I have disposed of some opinions regarding the alleged presence
of a second acid in sarkolactic acid already in 1877 (compare my
^ Pathology of the Urine,' 1877, p. 461 et seq.). I now prove for
the brain, what Erlenmeyer has proved for the flesh, namely, that
it contains only one lactic acid.
2. Formic Acid.
The acids extracted by ether after precipitation of the alkaloids
were in one experiment placed in a retort, and subjected to dis-
tillation. The main part of the distillate consisted necessarily of
acetic acid. The acids were neutralised by barium carbonate
and evaporated to crystallisation; the first crystals gave 53-09
per cent. Ba (acetate requires 53-72 per cent. Ba) ; the second
crystals gave 55*53 per cent. Ba, -and gave a strong formiate
reaction with nitrate of mercurous oxide. It is, therefore, pro-
bable that the water extract of the brain contains a small amount
of formic acid, as has already been stated by Von Bibra and
Mtiller. The amount was, perhaps, not greater than that of
succinic acid, to be described.
3. Succinic Acid.
{a) In Ox-brain. — The mother-liquors of the zinc lactate gave
with ferric chloride, not in the cold, but on boiling, a rust-coloured
precipitate, soluble in excess of chloride, forming a dark-red solu-
tion. The united mother-liquors were cautiously precipitated
while boiHng, and the compound filtered off. They were next
boiled with barium carbonate, and the precipitate filtered off".
U
210 CHEMICAL CONSTITUTION OF THE BRAIN
The latter was extracted with hot dilute acetic acid, in which
the ferric precipitate was insoluble. The ferric precipitates were
dissolved in water, and HoSO^, and extracted with ether; the
ether solution left the acid free.
The acid, easily soluble in water, crystallised on evaporation,
fused, and sublimed in white vapours. The vapours had a pun-
gent smell, and formed white crystals on condensation. The
acid was entirely volatilised without leaving any charcoal.
The sublimated crystals dissolved in water, and gave a clear
and colourless solution. This was cautiously neutralised by sodic
carbonate. The solution gave a rusty jorecipitate with ferric
chloride, soluble in excess of chloride. It gave a white precipitate
with mercurous nitrate, not altered by boiling ; gold chloride gave
no reaction, which excludes malonic acid ; uranic nitrate gave no
reaction. After boiling and concentration the solution of the
sodium salt gave a precipitate with BaCl^. This was soluble in
HNO.3, and reprecipitated by NH^HO ; still more by a little spirit.
This acid is consequently siccclnic, O^HgO^.
(b) In the Human Brain. — The process described in the fore-
going regarding the brain of the ox was repeated on the mother-
liquor of zinc lactate from the brain of man, and exactly identical
results were obtained. The ferric salt was decomposed and the
acid extracted by ether. It remained in a crystalline state, and
was sublimed. The reactions were then made upon the sublimate,
and found to agree exactly with those of succinic acid.
This experiment was repeated on a second quantity with iden-
tical results.
Succinic acid is thus shown to be a normal ingredient in small
quantity of the brain of man and of the ox. Miiller, when extract-
ing lactic acid from brain, had searched for succinic, but had not
obtained any. This is explicable on several grounds : firstly, his
method was not calculated to obtain it (he waited for crystals to
form in the concentrated lactic acid) ; and, secondly, the quan-
tities of brain-matter employed by him were probabl}^ too small.
The significance of succinic acid in nerve-marrow is probably
connected with that of the disintegration of the albuminous sub-
stances. But the possibility of merely accidental presence must
not be lost sight of, as succinic acid is present in many kinds and
parts of vegetables used for food by man and animals, and in
v/ine and other fermented liquids, in which it is produced by fer-
mentation from sugar.
ALBUMINOUS PFJNCIPLES. 211
YI.
GROUP OF ALBUMINOUS PRmCIPLES.
The brain as a whole is an aggregated mass of bioplasm, which
derives its peculiarity mainly from specific chemical additions.
The latter have been treated of in the earlier parts of this treatise,
and there remain for consideration the albuminous substances
which constitute the stroma of the bioplasm, in which the specific
matters are distributed, or with which they are combined in such
a manner as to produce the living brain-tissue or neuroplasm.
It is naturally deposited in the shape of cells and fihres, the cells
being termed ganglionic, because they were first observed in
nerve-ganglia, anatomically so-called. The fibres contain more
of the specific principles than the cells, but after they are de-
ducted, the albuminous principles in cells and fibres are very
much alike. By analytical means some part of each of the prin-
ciples can be extracted and identified, but it is as yet impossible
to separate the whole of them from each other unchanged. It is
found, principally by the method of extraction of the comminuted
tissue by means of salt water of varying concentration, that
neuroplasm contains small quantities of soluble albumen, partly
exhibiting the properties of serum albumen ; small quantities of
fibrin, and considerable quantities of what, from its similarity to
the body forming the stroma of the blood-corpuscles, has been
termed globulin, but which by its function is characterised as a
plastin. And in order not to assume that the plastin of the nerves
is identical in every respect with the plastin of other organs, I
propose to treat of it as neiiroplastin. In the process of extraction
of the five groups of constituents of the' brain, the albuminous
matters all become insoluble, and change their reactions with
those agents in which they were previously soluble. The
coagulated mass of brain-tissue after exhaustion with alcohol is
14—2
212 CHEMICAL CONSTITUTION OF THE BRAIN.
thus a mixture of curdled albumen, and of fibrin and neuroplastin
changed by heat and the influence of alcohol. It contains, further,
all the cytophosphatides from the nuclei of the nerve-cells, and
from the sheaths of the nerve-fibres ; the material of these sheaths
themselves, which is probably analogous in composition to that
of the sheaths of the muscular fibres ; and the tissue of the
capillaries, which pervade the brain-tissue. But neuroplastin is
by far the greater portion of the insoluble residue, and albumen
and fibrin do probably not amount to one-eighth of the weight of
the neuroplastin. The total weight of the albuminous matters of
the human brain, free from its membranes, amounts to 7*0 per
cent, at least; in some parts it is 7 '6 per cent., and may vary be-
tween these figures in different parts. Grey neuroplasm contains
7 '6 per cent., white tissue 8*6 per cent. This. is not quite equal
to half the solids in grey neuroplasm, which amount to about
15 per cent., while in white neuroplasm the amount of solids
rises much higher. The specific ingredients of white neuroplasm
may amount to 19*16 per cent., and if 8 "6 per cent albuminous
matters are added to this, we have a total of 27*76 per cent, of
solids, to which some salts have yet to be added. It is therefore
not surprising that some specimens of white neuroplasm should
yield as much as 30 per cent, of solids. The albuminous matters
have sometimes been found as high as 10 per cent., but it is
doubtful how far in these cases they were fully extracted with
alcohol. As much valuable information regarding the albuminous
substances in general had been obtained by chemolysis, even in
cases where the substances could not be obtained in a pure state,
I applied the process to neuroplastin in the first instance with the
following results :
Chemolysis hy Barita of Neuroplastin from Brain.
The albuminous matter was obtained by exhausting brain
(human or ox) with spirit of 85 per cent, strength.
An amount of neuroplastin about 100 g. in weight, with six
times its weight of crystallised barita and four times its weight
of water (part of the water being used to soak the albumen for
some time previous to the admixture of the barita), was taken for
the chemolysis.
The apparatus employed was an autoclave of wrought-iron, and
holding about 5 litres. The cover was air-tight and secured by a
ALB UMINO US' PRINCIPLES.
213
screw clamp, while an adjustable valve provided for the escape of
gas in case of the pressure rising beyond certain limits. The
mixture above described was placed in the chemolyser, and the
temperature raised to 180° 0. hy means of a gas-burner placed
underneath. The heat was maintained at this height for six
hours, whereupon the chemolyser was allowed to cool. When
the autoclave was opened, its contents emitted a strong smell due
to ammonia, compound ammonias, albuminol, and other products
of decomposition.
The semi-solid mass was extracted, placed in a platinum-still
with much water, and subjected to distillation. Two litres of
distillate were drawn off, containing ammonia and compound
ammonias in solution, and albuminol in small white flakes ; the
latter were removed by extraction with ether.
The ether solution on evaporation left a small residue consisting
of albuminol mixed with a peculiarly smelling, probably sulphurised
tody; the latter was gradually volatilised on standing, leaving the
albuminol, which crystallised.
The watery distillate was neutralised with hydrochloric acid and
evaporated to dryness, and the mixture of salts was further
treated for the separation of compound ammonias from simple
ammonia, as will be described below.
The mixture from which the volatile alkalies had been distilled
was filtered, and the insoluble matter isolated.
This insoluble maUer was treated with water, ether, and hydro-
chloric acid. A small quantity of a fatty acid went into solution
in the ether, barita with phosphoric and oxalic acid dissolved in
the acid water, while barium sulphate remained insoluble.
The soluble matters filtered from the foregoing precipitate were
freed from excess of caustic barita by crystallisation. From the
filtrate all barita was removed by excess of sulphuric acid, and
from the solution all acetic acid was expelled by distillation.
From the cold acid liquid alkaloids were removed by the addi-
tion of phospho-wolframic acid as long as a precipitate was
produced. This precipitate was washed with water containing
5 per cent, of sulphuric acid, and then decomposed with barita.
The solution containing the mixture of alkaloids was further
treated, as will be described below.
The acid filtrate from the phospho-wolframates, containing the
amido-acids, w^as freed from phospho-wolframic and sulphuric acid
2U CHEMICAL CONSTITUTION OF THE BRAIN
by barita, and evaporated slowly to crystallisation. It deposited
first tyrosin, then a mixture of leucin and tyrosin, then leucin
and small quantities of other amido-acids. Then a syrupy mass
remained, which contained yet some alkaloids, besides amido-
acids. Then alkaloids were as far as possible removed by a re-
petition of the phospho-wolframic acid precipitation just described.
Erom the liquid more leucin was obtained. At last there re-
mained a small quantity of uncrijstallisahle matter, w^hich was
treated as follows :
It was precipitated b}^ mercuric nitrate and sodium carbonate.
The hdhj ]jrecij)itate was washed with water by decantation, etc.,
and decomposed by hydrothion. The solution on evaporation
left a colourless gummy mass of the same character as the original
matter. It will be further treated of below. The mother-liquor
of the mercury precipitate was also freed from mercury, and
found to contain but little organic matter.
: The mixtures of tyrosin and leucin were united and warmed with
w^ater containing 10 per cent, of absolute alcohol. This dissolved
leucin, and left tyrosin undissolved. The limits of accuracy of
this process will be indicated lower down.
, Tyrosin was purified by solution in hydrochloric acid, treatment
with animal charcoal, and precipitation by sodium acetate ; the
precipitated tyrosin was crystallised from ammonia.
Leucin w^as purified by combination with copper, its solution
being mixed and heated with copper acetate in a manner the
details of which will be described below. In the course of this
23rocess a new leucin, the first isomer of the leucin hitherto
known, . was discovered, and from its sweet taste was named
glycoleucin.
The copper-compounds of the leucins are almost insoluble in
cold water, more soluble in boiling water. The copper-compounds
of other amido-acids which accompany the leucins seem all to be
more soluble in cold w^ater, particularly those of lower atomic
weights than leucin.
Glutaminic and asparaginic acid, which have been obtained by
chemolysis from other albuminous substances, have not yet been
isolated from the products of chemol3'Sis of brain-albumen. It
remains to be seen whether the compound alkaloids w^hicli have
been observed Avill on long-continued chemolj'sis yield these acids.
At present it seems as if the l»arita process applied to brain.
ALBUMINOUS PRINCIPLES.
215
albumen did not produce them. It is, however, not impossible
that they may be formed by the hydrochloric acid and tin process,
which evolves them easily from casein.
Nitrogen as Ammonia from Neuroplastin. — Barita chemolysis of
1 0 g. and distillation gave volatile alkali, which was neutralised
by hydrochloric acid, and combined with platinic chloride. The
dry salt was redissolved, and left 0*0550 g. insoluble matter,
while the soluble double salt of ammonio-platinic chloride
amounted to 2-8760 g. This corresponds to 0*1803 g., or 1*803
per cent. N. The insoluble platinum salt contained 0*0077 g. N.
This quantity of nitrogen is far below that obtained by the same
j)rocess from other albuminous matters. It approaches that ob-
tained from gelatin — namely, 2*55 per cent. It is less than
one-fourth of the total quantity of nitrogen contained in the
albuminous substance.
Total of Insoluble Barium Salts obtained from 10 [/. Neurojjlastin. —
These salts dried at 110°, weighed 4*1760 g., and on analysis were
found to have the following constituents :
Barium sulphate -
0*083
Barium _ . . . -
2*-425
Phosphoric acid (PO^)
• 0*099
Oxalic acid _ _ . .
0*043
Sulphurous acid - . . .
0-008
Hydrothion . . . .
trace
Carbonic acid and soluble organic matter -
1*246
Organic insoluble matter -
0*272
Total
4*176
In the foregoing experiment about 1 per cent, of phosphoric
acid was obtained.
Barium retained by Amido- Alkaloid- Acid Mixture. — The mixture
of amido-acids, alkaloids, and acids from which all excess of
caustic barita has been removed by carbonic acid, retains a con-
siderable amount of barium in solution ; this has to be removed
previous to the distillation of the volatile acids. The amido mix-
ture from 10 g. of albumen yielded 1*7830 g. barium sulphate,
equal to 1*0480 g. Ba, or 10*48 per cent. This is only about half
the quantity of barium retained in the chemolytic products of
other albuminous substances, ossein excepted, in the products of
which the barium retained amounts to 13*2 per cent.
216 CHEMICAL CONSTITUTION OF THE BE A IN.
Quantation of Acetic Acid from Neuroplastin. — The acid distil-
late from 10 g. of brain-albumen, chemolysed as above described,
yielded 0-1586 g. barium salt (BaC^H^jO^, At. W. ^ 255) equal to
0*074 acetic acid, or 0*74 per cent. This is only one-fourth of the
quantity yielded by pure albumen, and only half that yielded by
ossein or ichthyocollin.
Quantation of Tyrosin from Neuroplastin. — The tyrosin obtained
by the chemolysis of 10 g. of substance was separated by alcohol,
recrystallised from ammonia, and dried at 105°. It weighed
0'122 g. = 1-22 per cent. This is the smallest percentage of
tyrosin as yet obtained from any albuminous substance by this
process. Tyrosin is retained by the amido-mixture, and even by
crystallised leucin. The quantities of tyrosin actually isolated
must therefore be considered as minima until absolutely reliable
methods for its isolation may have been found out.
Leucins from 100 g. of Ox-Nenroplastin. — They were combined
with copper by solution in boiling water and addition of excess
of copper acetate. On boiling, a precipitate ensued, which was
filtered off and washed with cold water until the washings were
no longer blue. This precipitate was now boiled with much
water, and the blue solution was evaporated to dryness. The
residue from this evaporation is product No. 2, while the salt
which remained insoluble in boiling water {i.e.^ in as much as was
applied) constitutes product No. 1. The mother-liquor of the
first precipitate was evaporated as long as precipitates Avere
formed. These constitute product No. 3.
Quantation of Copper in the three Froducts.
No. 1 contained - - - 19*05 per cent. Cu.
No. 2 „ - - - 20-26 per cent. Cu.
No. 3 „ - - - 19-5 per cent. Cu.
The latter amount of copper is almost that required by cupric
dileucin. The solution obtained by decoction of the first preci^^i-
tate contained probably some amido-acid of lower atomic weight,
which raised the amount of copper to 20-26 per cent. ; while the
residual salt probably contained an amido-acid of higher atomic
weight than leucin, whereby the amount of copper was dei)ressed
to 19*05 per cent. These hypotheses will have to be further
tested on larger quantities of material.
ALBUMINOUS PBINCIPLES.
217
Organic Body from Mercuric Chloride and Soda Precipitate.
The white precipitate was decomposed with hydrothion, and
the solution left a semicrystalHne substance. This was insoluble
in spirit of 85 per cent, strength. In its watery solution phospho-
wolframic acid produced no precipitate, showing that it was not
and did not contain any alkaloid. Alkaline copper solution on
application of heat gave no reaction. Ferric chloride produced
a brown precipitate, soluble in excess of chloride, with an intensely
red colour. Ammoniacal silver solution gave no precipitate, but
on boiling it deposited reduced silver. Copper acetate gave no
precipitate even on addition of alcohol. Acetate of lead gave a
precipitate only after the addition of much alcohol, and this
compound seemed to be an alcoholate. When dry it was found
to contain 5 7' 8 per cent, of lead. It was therefore probable that
the body was not and did not contain either glutaminic or aspara-
ginic acid.
Properties of Common Tasteless Leucin and of its Copper-compound,
studied with a view of establishing its Diagnosis from its Isomer
Glycoleucin.
Some tasteless leucin, made from albuminous matters (not
brain) by the sulphuric acid process, was treated with cupric
acetate, and the first precipitate was removed. (In this any
glycoleucin would have been contained.) The filtrate from the
first precipitate on evaporation gave a precipitate (termed ' first
precipitate by evaporation,' being second precipitate of copper
process), which was decomposed by hydrothion. The solution, on
concentration, gave a first crop of crystals, which were rhombic
plates, and quite tasteless.
This was again combined with copper, and the precipitate was
filtered off. The precipitate was now boiled with water re-
peatedly until it was completely dissolved, and the solution was
filtered hot.
Quantations of Sohdnliti/.
(a.) 730 cc. saturated boihng deposited, mainly immediately
after filtration, and only to a small extent during cooling and
standing for twenty-eight hours, blue light crystals, which when
dry, weighed 0'1790 g., indicating a deposit of 1 part out of
4,078 parts of boiling water.
218 CHEMICAL CONSTITUTION OF THE BRAIN
(h.) Of the clear blue filtrate from the foregoing operation
630 cc. were evaporated to dryness, and left 0-1048 g. of blue
compound, showing that 1 part was soluble in 6,011 parts of
cold water.
(c.) 100 cc. on evaporation left 0*0162 g. residue, equal to a
solubility of 1 part in 6,172 parts water.
IiestiUs: 730 cc. of the leucin copper solution contained while
saturated at the boiling heat 0-3000 g. of salt; of this, 0'179 g.
was deposited on cooling, while 0-1210 g. remained dissolved at
the ordinary temperature. The solubility of the ' salt in boiling
water is therefore 1 part in 2,433. This is about double the
solubility of glycoleucin copper.
It is generally stated in chemical writings that leucin dissolves
in 27 parts of water at the ordinary temperature. I find by
special experiment made with pure tasteless leucin, that 1 part
dissolves in 30 parts of water at 15° C. (Of glycoleucin, 1 part
is soluble in 82 parts of water at 18° C.) It requires 658 parts
of spirit of 75 per cent, strength for solution. Combination with
co2)per therefore reduces its solubility very greatly.
The immediate deposition of crystals from a saturated leucin-
copper solution filtered hot indicates the presence in the com-
pound of ordinary or tasteless leucin ; the hot saturated solution
of glycoleucin-copper differs from the former in this, that it forms
a deposit only after long standing.
StahiUti/ and Begularity of Composition of Leucin Copper Compound.
A quantity (2-3 g.) of crystallised ordinary tasteless leucin, of
similar origin as the foregoing, and separated from copper with
which it had been combined, was a second time converted into
the copper compound by means of copper acetate. The compound
was extracted several times with boiling water, and then dis-
solved entirely in a sufficiency of boiling water. Soon after
filtration there separated from the hot solution a cnidcdUnc
deposit, which increased only slightly while the liquid cooled to
the ordinary temperature of the air. The precipitate was isolated
and dried at 110°, and contained 19*15 per cent. Cu. The solu-
bility of this precipitate in boiling water was as follows : 200 cc.
of boiling solution saturated by long boiling with excess of salt
left' 0-0904 residue, equal to a solubility of 1 part in 2,212 parts
ALBUMINOUS FFJNCIPLES.
219
of boiling water. The cold solution filtered from the precipitate
on evaporation left a residue, which showed that 1 part had
been dissolved in 5,882 parts of water.
The soMo?i in which the crystalline deposit, described in the
foregoing, had been formed, was evaporated to a small bulk as
long as it formed any deposit while hot, and then allowed to
cool. The isolated (here termed ' second ') precipitate dried at
110°, contained 19-57 per cent. Cu. 200 g. of cold water dissolved
0'0432 g. of this compound, which is equal to a solubility of 1 part
in 4,630 parts of water.
The filtrate from the second dejjosit just described was evaporated
to dryness to obtain the remainder of the leucin compound. Some
copper compound, differing in appearance from the leucin com-
pound, was deposited with the latter, and had to be removed by
water acidulated with a little acetic acid. This, as the quantation
of the copper contained in the compound showed, succeeded onl}^
partially, for it still contained 20-39 per cent. Cu.
Thus it will be seen that the very first crystals of this prepara-
tion (reconstituted a second time as copper-salt, after having been
constituted the first time as copper salt, with an apparently pure
specimen of leucin) contained a little less copper than is de-
manded by theory ; the second crystals, obtained by concentra-
tion, yielded the theoretical amount of copper ; and the third
fraction was evidently impure by the admixture of a copper salt,
which was perhaps a combination of an amido-acid of lower atomic
weight than leucin.
In connection with this it deserves to be mentioned that the
presence in leucin of tyrosin as an impurity depresses the amount
of copper which will be found in the copper compound made
from such leucin. 11*2 g, of crystallised leucin were treated in
boiling watery solution with copper acetate, and the deposit was
collected. It gave on analysis (mean of two quantations) 17-25
per cent. Cu. The filtrate on further evaporation and cooling
gave another deposit, which on analysis was shown to contain
18-18 per cent. Cu. Another 11-2 g. of the same leucin as that
used in the experiment just related, was dissolved in a large
quantity of warm water, and allowed to cool slowly, when it de-
posited a sensible portion of tyrosin. The leucin was now removed
by the copper process, and the compound found to yield the theo-
retical amount of copper. The mother-liquors of the first and the
220 CHEMICAL CONSTITUTION OF THE BIIAIN
second part contained a copper salt of a lower amido-acid, which
had a sweet taste, but was not glycoleucin.
Note. — This and similar copper salts, not yet exactly identified,
retain tyrosin in solution. When the copper is now removed by
hydrothion, the filtrate, reduced to the same bulk as the original
solution, deposits tijrosin. This first deposited tyrosin gives a
good mercurous nitrite test, but may contain some of the following
neiu hody. AVhen the mother-liquor of the foregoing tyrosin is
slightly evaporated, it deposits a body much like tyrosin, crys-
tallising in microscopic fine needles, but giving no mercurous
nitrite reaction ; a slight rose-pink which appears at the moment
when the reagents are mixed, disappears, and the mixture becomes
white.
Leucin obtained hy Crystcdlisatmi from Amido-Mixture from Clie-
mohjsis of Neurojjlastin. — Although this leucin had no distinctly
sweet taste, it was treated with copper acetate to separate any
glycoleucin which might be contained in it. It was dissolved in
hot water and heated to boiling ; a concentrated solution of copper
aceta.te was now added gradually until a precipitate was formed.
This first p'ecqntate was removed by the filter. To the filtrate
was added copper acetate as long as a precipitate was produced,
and then a slight excess of the acetate. The mixture was allowed
to stand and deposit the second preciintate. This was collected on
a filter. The filtrate was evaporated to a small bulk, and deposited,
particularly on cooling, a third j^rodiid. This latter appeared the
most crystalline, and of the deepest blue colour.
The first ^precipitate was then boiled seven successive times with
large quantities of water to extract the common leucin copper
compound, and leave any glycoleucin copper compound insoluljle
behind. It was then dried on the filter ; lastly in the air-bath at
110°, and analysed; it contained 19-60 per cent. Cu, or exactly
the theoretical amount of cupric dileucin.
The secoml precipitate was boiled with nine successive large
volumes of water, and what remained undissolved was analysed ;
it contained 19-49 per cent. Cu.
The third precipitate which crystallised out of the concentrated
mother-liquor of the first and second precipitate, was finely tritu-
rated in a mortar, washed with cold water, dried at 110^, and
analysed ; it contained 19-60 i)er cent. Cu (again exactly the tlieo-
I'etical amount required hy cupiic dileucin).
ALBUMINOUS PRINCIPLES.
221
The first and second precipitate were united, suspended in
boiling water, and decomposed by a current of hydrothion. The
bulk of the copper-sulphide was removed by filtration ; the hazy
and coloured liquid was evaporated to collect and precipitate some
copper-sulphide (which is frequently imperfectly precipitated from
neutral organic liquids), and after final filtration evaporated to
crystallisation. The latter process was aided by the addition of
some strong alcohol. The crystals were collected, and recrys-
tallised from water and alcohol and dried in the air-bath. On
elementary analysis they yielded results agreeing closely with the
theory of pure leucin.
Glycoleucin, the first Chemohjtic Isomer of Leucin, its Properties and
Combinations.
Glycoleucin has been obtained by me in two ways, namely,
synthetically, and by the chemolysis with barita of brain albumens.
I have not obtained it from animal albuminous matters by the
sulphuric-acid process, which yields ordinary tasteless leucin.
The mode in which it is separated is the following : The
mixture of the several leucins, obtained by crystallisation from
the amido-mixture, is combined with coj^per, by treatment with
acetate. The glycoleucin copper compound is mainly deposited
with the first part of the precipitate ; later, a mixture is deposited ;
lastly, mainly the compound of ordinary leucin. The copper
compounds which result are then exhausted with boiling water.
The glycoleucin copper compound, as the least soluble, remains
behind, and is ultimately almost ' insoluble in boiling water.
Much glycoleucin copper of course dissolves with the compound
of ordinary leucin. The latter can to a large extent be isolated
by the property that it separates from the boiling saturated solu-
tion in water almost immediately after filtration, while the
saturated boiling glycoleucin copper solution does not deposit the
excess of its salt until after long standing. It will be evident
that these processes can only yield small quantities of material
at a time. Seven specimens of glycoleucin copper Avere decom-
posed by hydrothion, etc., and crystallised separately. Three of
the crystallised products were subjected to elementary analysis,
and showed the identit}^ of their composition with that of leucin.
The seven specimens of crystals were then united (they weighed
7-53 g.) and dissolved in boiling water. To the boiling solution
222 CHEMICAL CONSTITUTION OF THE BRAIN.
a concentrated cold solution of cupric acetate was added, not in
excess. A sky-blue precipitate ensued, and was filtered off while
the liquid was hot. To the filtrate, while cooling, a further
quantity of cupric acetate was given, until no further precipitate
was produced.
Freci/pitate No. 1. — Dried at 110° contained 19-20 per cent. Cu.
Frecijpitate No. 2. — Dried at 110° contained 19 -45 per cent. Cu.
An eighth specimen of pure glycoleucin was combined with
copper, and the comj^ound on analysis gave 19 '45 per cent, of Cu.
The mother-liquor of this on concentration yielded a preparation
which on analysis was found to contain 19-4:2 per cent. Cu.
These data agree pretty closely with the theory of monocupric
dileucin, which contains 19'60 per cent. Cu = 2(CgHjoN02)Cu, or
C,2H,,CuN,0,.
Solubility of the Cupic Salt of Glycoleucin in Cold Water. — Pure
salt was boiled for a long time with water ; th^ mixture was
allowed to cool ; when quite cold the solution was filtered from
the compound. 200 cc. at 16-5° C. on evaporation left 0*0228 g.,
equal to a solubility of 1 part in 8,772 parts of water. The
solubility of ordinarj^ leucin copper is 1 part in 6,172 parts of
water, and is therefore greater than that of glycoleucin copper.
During the operation no trace of reduction or blackening was
observed, not even when the solution of the compound was eva-
porated to an extremely small bulk with excess of cupric acetate.
(This constitutes a diagnostic difference from the bearing of a
more soluble sweet product which is obtained from the mother-
liquors of the amido-mixture, and during the evaporation of which
with coi)per acetate such a reduction takes place.)
Solubility of the Cujpric Salt of Glycoleucin in Boiling TFater. —
200 cc. of the boiling solution left on evaporation to dryness
0-0449 g., or 1 part dissolved in 4,454 parts of boiling water.
This is about double the solubility of the salt in cold water. On
cooling, the solution becomes only slightly opaque ; a long time
is required for it to deposit the salt soluble in the hot as a visible
precipitate.
The glycoleucin copper compound crystallises in minute scales
and plates, combined in masses or balls. Many of the scales are
clearly rhombic, others rhoml^o-hexagonal.
Elementary Quantation of Glycoleucin, p-eparecl from Insoluble
Copi?er Salt. ■ -The copper was removed by hydrothion, and the
ALBUMINOUS PRINCIPLES. 223
glycoleucin obtained pure and pearl-white by repeated crystallisa-
tion. It was dried over calcium chloride, and at last at 110" C.
At. Wgts.
6 C 72
13 H 13
N 14
2 O 32
131 0-00
Glycoleucin does not give the inosite reaction with mercuric
nitrate.
Solubility in Water at 18°. — 100 parts by weight of solution
retain 1*22 parts of glycoleucin in solution, or in round numbers
1 part is soluble in 82 parts of water. Glycoleucin is therefore
much less soluble in water than common tasteless leucin, of which
1 part requires 30 parts of water for solution at 15°.
The sweet taste of glycoleucin is less easily perceived on the
crystals than on their saturated solution. Of this one drop will
give a distinctly sweet taste over a great part of the mouth. The
intensity of the sweetness is not much less than that of inosite.
A New Reaction and Compound of Cerebral Tyrosin.
I have shown that tyrosin is present amongst the amidated
bodies extracted from the brain, though in small quantities only.
It is also a product of the chemolysis of neuroplastin, and cannot
be separated so completely from the amido-mixture as is desirable,
and as is generally believed. To improve the processes of this
separation I have made some experiments which have resulted in
the following. The experiments were made on tyrosin from
neuroplastin.
Tyrosin dissolved in hot water with the aid of caustic soda ley,
on addition of mercuric chloride, gives a deep yellow solution,
and no precipitate. On cooling the mixture becomes turbid, and
on reheating a yellow precipitate ensues. The solution of tyrosin
must be dilute, hot, must contain excess of caustic ley, and the
i mercuric chloride must not be added in excess ; if excess be
added, a yellow precipitate is immediately produced. If to the
dilute, hot, alkaline solution of tyrosin mercuric chloride is added
Synojjsis of Theory and Data.
Found.
Percent. (a.) {b.) (c.)
54-96 54-92 — —
9-92 10-02 — —
10-69 — 10-61 10-72
24-43 _ _ _
224 CHEMICAL CONSTITUTION OF THE BBAIN.
gradually, until a faint yellow precipitate is produced, and the
solution is heated and allowed to stand, the slight precipitate
which forms on heating crj'stallises entirely in golden-brown
crystals, and is Hg.20Cl2. The yellow amorphous precipitate,
which forms out of the j^ellow solution on application of heat, is
entirely soluble in excess of caustic soda. On heating the solution
to boiling, no immediate precipitate is now produced. If the
heating and addition of mercuric chloride are done very gradually
near the boiling-point, until the yelloAv fluid becomes opaque, it
sets on cooling into a white solid jelly. This compound is dis-
solved in excess of caustic soda, the solution is filtered, and the
compound is then reprecipitated with acetic acid. It is then
jDerfectly white. The compound is therefore insoluble in weak
soda, soluble in great excess of soda, and insoluble in dilute
acetic acid. Dried over oil of vitriol in vacuo and analysed, the
compound gave the following data :
Found
Percents.
C 18-25
1- 78
2- 42
61-37
6-48
9-70
H
N
Ho
CI
O
Synopsis of Data and Theory.
Hypothetical
rormula.
N
•by At. Wgts.
1-520
1-780
0-1728
0-3069
0-1825
0-6062
-byN
8-7
10-3
1-
1.
1-7'i
1-
3-4
CI
0.
2 + HgO.
100-00
or 2(Cc)HioN03 + HgCl) + HgO, or expanded in the following
formula :
Hg-Cl
C„H,„N0,
I
C,,H„N03J
] HgO.
Hg-Cl
It will thus be seen that the reaction essentially consists in a
reduction of mercuric to mercurous chloride, which remains com-
bined with tyrosin ; and of this compound two molecles are
soldered together by a molecle of mercuric oxide. It is not
intended to fix the exact manner in which, and place (in the
molecle) at which this union is effected. The reduction of the
ALBUMINOUS PRINCIPLES.
225
mercuric to mercurous chloride is no doubt effected by that part
of the hydrogen of the tyrosin which does not appear in the })re-
cipitate ; in other words, an atom of hydrogen is substituted by
calomel. For this latter substitution the ordinary didynamic
character of mercury affords an easy opportunity, but for the
soldering action of mercuric oxide the metal may hypothetically
be allowed to possess a greater number of dynamicities than
two.
Mercuric chloride is a general reagent for bodies of the alkaloidal
class. In this capacity it produces precipitates in almost all animal
fluids which contain albuminous matters or their derivates down
to ammonia. The composition of these precipitates is as yet little
studied, but is complicated enough to have deterred inquirers
from ascertaining it. I have examined some such precipitates,
e.g. from the extract of the liver in kidney diseases. In such, the
reduction of mercuric to mercurous chloride is effected massivel}^,
but only a part of the calomel produced remains in combination.
The foregoing is a key to the study of such combinations.
New' Alkaloids oUained from Neuroplastm hj Chemohjsis.
The amido-mixture, which is obtained from neuroplastin by
decomposition with barita under pressure, yields on first crystal-
lisation leucin, glycoleucin, tyrosin, and some amido-acids of lower
atomic weight than leucin. But there remains a considerable
part of the mixture which does not crystallise in the state of
mixture, each ingredient of which, however, seems crystalline
after complete separation. I have isolated two of the ingredients,
and give in the following a short description of their isolation
and their properties.
Mode of Isolation of Alkaloids from Armdo-Mixture. — The mother-
liquor of leucin is diluted, acidified with sulphuric acid, and mixed
with an acidified solution of phospho-wolframic (syn. phospho-
tungstic) acid, as long as a precipitate is produced. The latter is
washed with water containing 5 per cent, of oil of vitriol, by
decantation, ultimately on the filler. It is then decomposed with
hot barita solution, the excess of barita is removed by carbonic
acid, and the resulting solution of alkaloids is evaporated to a
syrup. This product has the following properties and reactions :
It is strongly alkaline ; has the smell of sperma ; is soluble in
15
226 CHEMICAL CONSTITUTION OF THE BRAIN.
ammonia water ; does not reduce potassio-cupric tartrate, ^^lth
hydrochloric acid and gold chloride it gives a copious precipitate,
which is insoluble in excess of acid, easily soluble in alcohol.
Hydrochloric acid and platinic chloride give no precipitate in
watery solution, but in alcohol a precipitate is produced. Zinc
chloride gives a copious white precipitate, which is soluble in
excess of the chloride and in hydrochloric acid. Silver nitrate
gives a voluminous white precipitate, soluble in nitric acid. Tannin
gives a voluminous white jorecipitate. Mercuric chloride produces
a striking phenomenon. When to its saturated solution a drop of
alkaloidal matter is added, the whole surface is instantly covered
with a white precipitate. (The matter precipitated is therefore
not spermatin, which gives only a turbidity with mercuric chloride.
Ann. Chem. Med. i. 306.) The alkaloidal matter is carbonated,
as on addition of an acid carbonic acid is evolved. It also
contains some barium not precipitable by carbonic, precipitable
by diluted sulphuric acid.
Separation of the Alkaloidal Matter into two Croups ly Absolute
Alcohol. — When the syrupy mixture of alkaloids is treated with
absolute alcohol, a viscous matter remains insoluble, another
j^ortion dissolves in the alcohol. The matter insoluble in alcohol
retains its solubility in water. The matter soluble in alcohol
gives with platinic chloride a copious precipitate of a double salt.
This is not changed by alcohol, but is altered quickly by water.
It fuses after evaporation of the alcohol. Placed in water it
practically dissolves, a part remains undissolved. The solution
in water continues to form deposits, for weeks, of the insoluble
salt. Owing to this lability I have not examined these two
platinum salts any further.
From the viscous matter precipitated by alcohol there was.
obtained by a process of continued crystallisation a white crys-
tallised alkaloid, very soluble in water, easily soluble in hydro-
chloric acid. The hydrochlorate evaporated to dryness, and
redissolved in water, crystallises from concentrated solution.
From spirit this hydrochlorate crystallises still better. The spirit
solution may be mixed with ether without yielding a precipitate.
But the crystals of the hydrochlorate are not very soluble in
ether and may be washed with it. The salt, mixed with mercuric
chloride and then with caustic soda, gives a white precipitate
soluble in excess of the soda.
ALBUMINOUS PRINCIPLES.
227
Eleriientanj Quantation of this neiv Alkaloid. — After removal of
the hydrochloric acid the free body crystallises in balls of needles.
Dry at 110°.
The analyses lead to the following provisional theory :
Found Percents. -^At. Wts. -^N = l.
C 52-99 4-4158 6-3
H 9-03 9-03 12-9
N 9-896 0-7068 1-
0 28-084 1-755 2-5
100-000
These relations of elements remind of a leucein (rather than a
leucin), with which probably a small quantity of a higher homo-
logue is mixed.
Separation of the Alkaloidal Matter into tico Groups hy Cupric
Acetate and Absolute Alcohol.
When the mixture of alkaloids is dissolved in little water and
mixed with a saturated solution of cupric acetate and warmed, no
precipitate of leucin copper ensues if the process of precipitation
by the phosphotungstic acid has been correctly conducted. On
continued warming some brown cuprous and dark cupric oxide
are deposited. The deep blue solution filtered from all deposits
is mixed with absolute alcohol as long as this agent produces a
precipitate. This is the copper compound of a new alkaloid,
which, without further purification, on analysis gave the following
preliminary results :
Synojms of Result and Theory.
Percents. -i-At Wgts. -^Cu = l.
C 38-23 3-186 12-95
H 5-91 5-91 24-
Cu 15-60 0-246 1*
N 10-61 0-757 3-
0 29-65 1-853 7-53
100-00
Leading to formula C^.^H^sCuNgO^, or Ci2H23N30g + CuO. The
salt has a very light blue colour ; it is easily soluble in water, and
the solution has a very dark blue colour.
15—2
228 CHEMICAL CONSTITUTION OF THE BRAIN.
The alkaloids which are soluble in absolute alcohol in the
presence of cupric acetate have not yet been isolated. They con-
tain, of course, the bodies which give the platinum compounds
above described. I have subjected them to numerous reactions,
and have isolated some peculiar bodies. But they must be pro-
duced in quantity before they can be studied with advantage.
These alkaloids were not observed by Schiitzenberger in the
researches on the chemolysis of various albuminous substances
of which I have given an abstract in Ann. Chem. Med. i.,
pp. 20-44. They will, therefore, have to be added to the list of
terminal cleavage products.
INORGANIC PRINCIPLES.
229
YII.
GROUP. OF INORGANIC PRINCIPLES, OR MINERAL
ACIDS, BASES, AND SALTS.
The ash of the fresh grey tissue of man amounts to about 1 per
cent. ; that of white tissue is about 1 '7 per cent. ; the same relations
are observed in the grey and white tissues of the ox. From the
former data it is apparent that an ordinary human brain contains
from 18 to 20 g. of ash or incombustible residue. This residue con-
sists, however, to the extent of about 48 per cent, of phosphoric
acid, of which about one-fifth is in the free state, while four-fifths
are in combination. The greater part of this phosphoric acid is
unquestionably derived from the phosphatides, and has replaced
nearly all the more volatile acids, such as carbonic, hydrochloric,
and sulphuric acid. But even so the phosphoric acid included in
the 1 "7 per cent, of incombustible matter is certainly less than one-
third of the phosphoric acid which is present in the phosphatides.
It is therefore clear that a great portion of the phosphorus present
in brain is volatilised in the course of the ordinary method of com-
bustion. This method, therefore, actually leaves only the bases
in an approximately complete manner. 100 parts of acid brain-
ash contain 32 parts of potash, 11 parts of soda, and some lime
and magnesia, for which we do not give figures, because we have
none of our own, but know that those given by others are in
correct. The analysis of the mineral ingredients of the brain
will therefore have to be resumed in the manner indicated in
the chapters which treat of the purification of the phosphatides,
the cerebrosides, and the final water-extracts. These latter, we
know already, contain sodium chloride, and sulphates, and phos-
phates, besides salts of organic acids. The cerebrosides and the
230 CHEMICAL COKSTITUTION OF THE BRAIN.
phosphatides remaining mixed with them have a great tendency
to retain potash, in a form which is not precisely known. The
phosphatides retain salts and oxides, amongst them lime, as
shown in a previous chapter. Future analyses for the mineral
salts will therefore have to be applied to four different materials,
the neuroplastin, the phosphatides, cerebrosides and cerebrinacides,
and the ultimate water-extracts.
QUANTATION OF CONSTITUENTS.
231
YIII.
QUANTITATIVE RELATIONS OF THE IMMEDIATE
PEINCIPLES AND CONSTITUENTS OF THE BEAIN.
A. QUANTATION OF THE CHEMICAL CONSTITUENTS OF
GREY TISSUE FROM THE HUMAN BRAIN.
Grey tissue was careful!}^ cut from the surface of both hemi-
spheres, and from the anterior and posterior lobes of each.
6 '2 395 g. were dried at 95° until the weight was constant. The
substance was repeatedly cut up with a knife. The dry residue
weighed 0*9193 g. = 14-73 per cent. The water lost amounted to
5-3202 g. = 85-27 per cent.
For the following operations another quantity of grey tissue,
amounting to 46 g. fresh, was cut from all parts of the brain. It
was extracted five times with boiling spirit in a flask attached to
a reflux cooler.
The albuminous residue^ dry, weighed 3-5 g., equal to 7-6 per
cent, of the fresh grey tissue. It wus analysed for sulphur and
phosphorus. 1*5304 g. were burnt with addition of barita-water,
and gave 0-0024 BaSO^= 0-02 per cent, sulphur, and 0*0325
magnesium pyrophosphate = 0-60 per cent, phosphorus.
The deposit which the spirit made on cooling (cerebrins, etc.)
weighed 0*3 g.
The spirit solution, evaporated to half its bulk, gave a (second)
deposit which weighed 0*1 g.
The filtrate from this was evaporated to a small volume, and
deposited a (third) deposit weighing 0*7 g.
The foregoing deposits (0*3 + 0-1+0*7, total =1*1 g.) were
extracted with ether. There remained 0*2 insoluble, 0-9 were
soluble in ether. The matter soluble in ether (previously de-
posited from spirit) amounted to 1*950 per cent, of the fresh
232 CHEMICAL CONSTITUTION OF THE BRAIN.
tissue, while the matter insohible in ether (cerebrius mainly)
amounts to onl}' 0434 per cent, of the tissue.
The last oily matter^ which could not be filtered from the water-
extract in which it was suspended, was precipitated with lead
acetate ; the curded precipitate was collected on a filter.
The irrecipitate of lead salts contained the phosphorised organic
compounds and sulphuric and phosphoric acid from the inorganic
salts. It was washed while being continuously stirred with water
to which a little lead acetate had been added. It was next ex-
tracted with absolute alcohol. The alcoholic solution contained
all the lecifhiii (with some lead). The dry matter weighed
0-73 g.
The insoluble in alcohol residue was completely dried and ex-
tracted with ether. The solution on distillation left all Jcejjhalin
lead, which weighed 0"22 g.
The insoluble in ether residue was extracted with boiling
alcohol, which dissolved matters which when diVj weighed 0"03 g.
The residue from the three extractions with alcohol, ether, and
boiling alcohol was mainly an inorganic lead salt. It was ex-
hausted with dilute nitric acid, and the insoluble residue was
recognised as lead sulphate. The matter soluble in nitric acid
was anal3^sed for phosphorus by ammonium molybdate, etc., and
gave 0-0049 P. T\\q ijliosphoric acid thus indicated was evidently
combined with alkalies in the grey tissue.
The lead salts from the mixture of the last oily matter and
watery extract obtained as just described are liable to contain
some inosite lead as an insoluble compound. When this is decom-
posed in warm absolute alcohol by hydrothion, an alcoholic
solution is obtained from which, on long standing, and again
after concentration, inosite is deposited in crystals. I have care-
fully identified these crystals, by their taste, their shape, and
their reaction with mercuric nitrate. It is therefore clear that
inosite can be precipitated, under the conditions prevailing in
this research, by neutral acetate at least in part. Its complete
precipitation can be effected only by hasir acetate, and that with
the aid of ammonia.
The v:aterij solution was freed from lead by hydrothion, con-
densed to expel acetic acid, acidified with sidphuric acid, and
extracted Avith ether. The extract gave by the usual treatment
zinc lactate in crystals, which weighed 0-07 g., equal to 0-04.52 g.
QUANTATIQN OF CONSTITUENTS.
233
lactic acid in 44 g. grey tissue, or 0-102 per cent. ; in round
numbers, one part of lactic acid in a thousand of grey tissue.
The solution from which lactic acid had been extracted was
treated with ammonium carbonate in excess, and gave clear
indication of the presence of calcium. The filtered solution was
boiled to expel ammonia, and then treated with phosphomolybdic
acid. A precipitate of alkaloids or extractives was obtained
weighing 0"530 g.
From the filtrate all phosphomolybdic and sulphuric acid were
removed by barita. The filtrate treated in the usual way and
evaporated to dryness left a residue, being inosife and carbonates
of alkalies weighing 0*69 g. It gave with mercuric nitrate the
inosite reaction. The residue was dissolved in water, and pre-
cipitated by lead acetate and ammonia. The precipitated inosite
lead was isolated and decomposed by hydrothion.
The mother-liquor of inosite lead was freed from lead, eva-
porated, burnt, etc., and left mixed carbonates of alkalies,
which were transformed into chlorides = 0-2560. Out of this
quantity platinic chloride precipitated 0-070 double chloride
= 0-0112 K = 0-0250 per cent. K in grey tissue. The remaining
sodium amounted to 0*0920 per cent, of grey tissue,
Synoims of the Besults of Analysis of Grey Tissue of Human Brain.
Percents.
Water expelled at 95°
- 85-270
Neuroplastin
- 7-608
Ether extracts, kephalin, and
lecithin
(and cholesterin "?)
- 1-950
Cerebrins and myelin
- 0-434
Lecithin \
Kephalin /■ from last oily -
Myelin j (and cholesterin ?)
- 0-780
Inosite - _ -
- 0-193
Lactic acid
- 0-102
Alkaloids
Sulphuric acid
traces
Phosphoric acid, H3PO4 -
- 0-017
Potassium
- 0-025
Sodium - - -
- 0-092
Water-extract
- 0-500
Loss in operations
The loss in operations is very large, amounting to almost a
quarter of the entire solids. It is mainly incurred through the
difficulty which there is of separating the last oily matter from
234 CHEMICAL CONSTITUTION OF THE BE A IN.
the matter soluble in water. But other operations equally involve
as yet unavoidable loss. The construction of suitable apparatus
for ether extraction would only partly avoid the loss by ether
extraction, as in this process the main difficulty is filtration.
B. QUAXTATIOX OF THE CHEMICAL CONSTITUENTS OF THE
WHITE TISSUE OF THE HUMAN BRAIN.
The parts to be analysed, amounting to 66 g., were cut from
the centre of the hemispheres and corpus callosum. They were
triturated to a pulp, mixed with spirit, and extracted six times
with boiling spirit.
The sinrit extract deposited a white cerehriii substance on cooling,
which was collected and washed. It was then extracted with
large volumes of ether and filtered again. It weighed, dried at
70", 4*5615 g., equal to 6-91 per cent, of the fresh white brain-
substance. Further treatment of the cerebrin bodies see below.
Ether extract of the cerebrin deposit. — The ether was distilled off,
the concentrated solution was filtered from a trifling deposit, and
distilled to dryness. The residue weighed 2-4150 g.
The ether extract of the albuminous substance (made after exhaustion
by spirit and drying) on concentration deposited a few oily drops,
and on distillation to dryness left a residue weighing 0*0350 g.
It became dark, and existed only as a minute brown coating on
the glass, possibly a mere trace of phosphorised substance, too
small in quantit}'' for further treatment.
The neurojplastin residue was dried and weighed, after the treat-
ment with ether, 5-70 g., equal to 8*63 per cent, of original
white brain-substance.
The spirit extract which had deposited the white matter was
concentrated twice in succession, and after each concentration
deposited a semi-crystalline buttery matter. Of this the ether
extract weighed 5-1730 g., while the part insoluble in ether
weighed 0*1400 g. Of this a part was soluble in boiling spirit,
and deposited on cooling, while a dark part was insoluble in boil-
ing spirit, and contained some inorganic matter and phosphorus.
The cerebrin mixture above described, weighing 4*5615 g., was
dissolved in boiling absolute alcohol, when 0*1815 g. of coloured
matter, which was insoluble in benzol, and therefore was not
stearoconote, remained undissolved, =0*275 per cent, of tissue.
(It amounted to 3*9 per cent, of the cerebrin mixture, and besides
some slight impurity was neuroplastin. )
QUA NT AT ION OF CONSTITUENTS.
235
The dissolved part deposited, on cooling and standing, phrenosin,
kerasin, cerebrinic acid, with some phosphorised matter, and
retained a phosphorised matter in solution. The dry cerebrins
weighed together 2-6030 (equal to 3*94, in round numbers
4 per cent., of the white tissue).
The alcoholic solution from which these cerebrins had been
deposited, measuring 750 cc, contained 1-770 g. of matter dis-
solved, being phosphatides, amidolipotides, cerebrosides and
cerebrinacides. With this some attempts at identification and
quantation were made. Out of one half, 375 cc, a precijjitate
was obtained with the aid of alcoholic platinic chloride ; the pre-
cipitate, dried at 65°, weighed 0-4524 g., and contained phosphorus
= 2-52 per cent. The second 375 cc. were precipitated with
alcoholic cadmic chloride; the precipitate weighed =0-2965, and
contained phosphorus = 4*78 per cent.
The cadmium salt therefore contained much more phosphorus
than the platinum salt ; in other words, they contained different
organic ingredients. Moreover, the precipitants brought down only
about one-sixth part of the matter in solution in absolute alcohol.
The mother-liquors were concentrated and made deposits, which
were examined, but the results were not of a nature to be quoted.
The substances which remain dissolved in much absolute
alcohol amount to 2-6 per cent, of the white tissue.
The last oily matter which was suspended in the aqueous extract
could not be separated by filtration. This is one of the greatest
difficulties of brain analj^sis, and will have to be overcome by
further discovery. In the interval I- have adopted the following
process, which is efficient in all directions except on the point of
inosite, as I have already stated above. The mixture was treated
with lead acetate ; the precipitate was dried (and weighed 0-7 340 g.).
It was extracted with cold absolute alcohol. (The extract weighed
0-4802 g.) It was mainly lecithin, with some lead.
The part insoluble in cold absolute alcohol was extracted with
ether ; the solution left a residue of kephalin lead, weighing
0-2030 g.
The part insoluble in ether was mainly myelin lead, but con-
tained some sulphuric and phosphoric acid. It weighed =0-1970 g.
The watery filtrate from the lead precipitate was freed from lead
by hydrothion evaporated to dryness, and left a residue 0*9260 g.
This was redissolved in little water, and acidified with sulphuric
236 CHEMICAL CONSTITUTION OF THE BUAIN.
acid. The mixture was extracted with ether, and the extract
treated for lactic acid, with zinc, etc. The zinc lactate crystallised
white, weighed =0*0707 g., dried over oil of vitriol in vacuo.
The acid solution from which the lactic acid had been extracted
was precipitated with phosphomolybdic acid, and the precipitate
filtered off and dried =0-1270 g.
The filtrate from this was treated with barita, etc., and after
treatment with ammonium carbonate and evaporation left a
residue weighing 0*7500. This was redissolved in water and
precipitated with basic lead acetate. Inosite lead w^as isolated
and decomposed with hydrothion. The dry inosite, crystallised,
weighed =0-1420.
The mother-liquor was freed from lead, evaporated to dryness,
burnt, and the residue weighed =0-171 g. ; of the metals in this,
17*67 per cent, were potassium, and 82*33 per cent, sodium.
Synopsis of the EesuUs of Analysis of White Tissue of Human Brain.
Water expelled at 95° - - - - 70-230
Neuroplastin - - - - - 8*630
Ether extracts, kephalin, lecithin, and cholesterin - 11*497
Cerebrins and myelin - - - - 6*910
Insoluble in ether from buttery - - - 0-212
Lecithin (lead) ----- —
Kephalin (lead) ----- —
Myelin (lead) - - - - - —
Water-extract, 1*403 per cent, consists of - - —
Lactic acid ------ 0*0456
Inosite - - - - - - 02151
Alkalies (carbonates) - - - - 0*1717
The separation of the ether extracts into their constituents was
also carried out, and the results will be stated lower down.
C. QUANTATION OF THE ABSOLUTE AND SPECIFIC GRAVITIES
OF A HUMAN BRAIN AND OF SEVERAL PARTS.
Absolute Weight
Division of the Brain. Weight in in
Air. Water.
1. Eight hemisphere - 589-035 20*820
2. Left iiemisphere - 595-823 21-600
3. Cerebellum - - 135-172 5-030
4. Mesenkephalon - 33*950 1*250
5. Sclerotic part - 3*630 0*150
Entire Brain - 1357-610 48-850 1308-700 1-037
Loss of cj 'c
Weight in Specie
Water, ^""^^'^y-
568'215 1*037
574*223 1*038
130-142 1-039
32-700 1*038
3-480 1*043
QUANTATION OF CONSTITUENTS.
237
Quantation of the Specific Gravihj of White Tissue and Grey Tissue
of the Humctn Brain.
These specific gravities were ascertained by suspension of the
parts in water, etc. (grammes at 16°).
Wgt. in Air.
Wgt. in Water.
Spec. Grav.
0-4870
0-0258
1-053
0-8650
0-0400
1-048
1-0746
0-0477
1-046
0-6859
0-0310
1-044
1-0479
0-0394
1-039
3-7845
0-1319
1 -036
9-7000
0-2850
1-030
9-5362
0-2740
1-030
Greij Tissue.
Wgt. in Air.
Wgt. in Water.
Spec. Grav.
0-6628
0-0243
1-038
14-4592
0-3810
1-027
11-5741
0-2865
1-025
The foregoing data have been arranged in the order of decreas-
ing sjDecific gravities. This led at once to an inverse order in the
column denoting weights in air. Only two figures out of eleven
do not absolutely take the places which they would occupy if the
order of increase in the first column was inverse as that in the
third.
It therefore appears, what has also been confirmed by many
other experiments, some to be related below, that the sjMcific
gravity of white and grey tissue of the brain is found the higher, the
smaller is the quantity of hrain-tissue employed in the experiment. Now,
as the pieces of tissue which can be employed in the experiments
are limited in size by the arrangement of the relative tissues
in the brain, it is clear that the specific gravity quantations
of brain-substance in water can only be approximately correct.
The variations, no doubt, depend upon a reaction between the
surface of the piece of tissue immersed and the water which sur-
rounds it. The water takes up some soluble albumen and some
salts, and the piece of brain-tissue immersed assumes a glazed
appearance. It is evident from this that the greater the surface
238 CHEMICAL CONSTITUTION OF THE BRAIN
of the piece under observation to its volume^ the greater will be
the effect of this source of error. The error will further be
influenced by the length of time during which the piece of tissue
is immersed, and consequently variations will arise, even when
pieces of equal size are examined, from the interference of the
accident of quicker or slower weighing. It is further doubtful
whether white and grey tissues will be equally influenced by
water in the same time, even when their bulks are equal. It is
further not j^roved that either the grey or the white tissue is so
homogeneous in any part of the brain as is assumed for the
purposes of comparison. It is therefore clear that specific gravity
estimates of brain-tissue in water have only an approximative
and no absolute mathematical value. Such estimates must there-
fore be made with the aid of fluids of well-known specific gravity,
which, while they make contact with the brain-tissue, do not
provoke in it any chemical or physical changes.
Quantations of the Sjmfic Gravities of White and Grey Tissue from
different parts of the Brain.
White Tissue of Hemispheres.
(a.) Quantations by the Piknometer :
Absol. Wgt. Subst. + Pik. Pik. + Water.
0-4526 61-9300 61-9120
0-2817 61-9342 61-9198
{b. ) Quantations hy Suspension in Water :
Absol. Wgt. Wgt. in Water. Spec. Grav.
0- 6859 0-0310 1-044
1- 0479 0-0394 1-039
3-7845 0-1319 1-036
9-5362 0-2740 1-030
Spec. Grav.
1-041
1-054
Grey Tissue of Hemisphere,
(a.) Quantation by the Piknometer:
Absol. Wgt. Subst. + Pik. Pik. + Water. Spec. Grav.
0-3117 61-9146 • 61-9039 1-039
(b.) Quantations by Suspension in Water :
Absol. Wgt. Wgt. in Water. Spec. Grav.
0-6628 0-0243 1-038
14-4592 0-3810 1-027
LOHDDh
QUANTA TlOJSr OF CONSTITUENTS, 239
TFhite Tissue of Cerebellum,
(a. ) Quantation hj Pihnometer :
Absol. Wgt. Subst. + Pik. Pik. + Water. Spec. Grav.
0-5900 61-9240 61-9039 1-037
{!).) Quantation by Suspension in Water :
Absol. Wgt. Wgt. in Water. Spec. Grav.
0-8650 0-0400 1-048
White Tissue of Mesenkeplialon.
(a.) Quantation by Piknometer :
Absol. Wgt. Subst. + Pik. Pik. + Water. Spec. Grav,
0-6849 61-9252 61*9039 1-032
(b.) Quantation by Suspension in Water :
Absol. Wgt. Wgt. in Water. Spec. Grav.
0-5128 0-0258 1-053
Synopsis and Averages of the Specific Gravities observed in the three
Series of Observations detailed in the foregoing, without reference
to repetition of the same numbers or to quantities on which they
were observed.
I. White Tissue.
1. 1-054 5. 1-044 9. 1-036
2. 1-053 6. 1-051 10. 1-032
3. 1-048 7. 1-039 11. 1-030
4. 1-046 8. 1-037
Mean specific gravity of white tissue = 1-041.
II. Grey Tissue.
1. 1-039 2. 1-038 3. 1-027 4. 1-025
Mean specific gravity of grey tissue = 1-032.
Specific gravity of entire brain (four parts) = 1*037.
The foregoing figures do not difi'er much from those accepted
by other observers. The specific gravity of white tissue is the
same as that commonly allowed in physiological treatises, namely
1-041, while the specific gravity of grey tissue is 1-032 instead of
that commonly allowed, namely 1-034. But when it is con-
sidered that what must theoretically be assumed to be the best
observations of the specific gravity of white tissue, namely those
on the largest volume, do only give 1 -030 as the value, while grey
240 CHEMICAL CONSTITUTION OF THE BRAIN
tissue under the same limitation gives 1*027, it is impossible to
avoid the suspicion that all specific gravity estimates hitherto
made, including the foregoing, are vitiated by a fundamental
fault of method, or by several faults, as above indicated. These
probable faults have for the first time been observed in the
course of the present researches, and there has been no time for
remedying the inconveniences arising out of the observation.
In 1876, I communicated a then new method for estimating
the proportion between white and grey tissue in the brain
(an important physiological problem, which seems anatomically
quite insoluble) by a calculation from the four factors,
absolute weight of the brain, specific gravity of the entire brain,
and specific gravities of each white and grey tissue. This
method still has its future, if the diff'erence between the specific
gravity of white and grey tissue be not ultimately found too
small.
In its execution the following formula may be used :
Vwip — q) . , . ,
X = — — -lii, m which
X = quantity of white m^atter.
P = absolute weight of the brain.
2^ = specific gravity of the entire brain.
g = specific gravity of the grey tissue.
to = specific gravity of the white tissue.
Applying this formula to the data given above, namely :
P = 1358; j> = l-037; g-im2 ; ic=l-OU ; then = 757-3,
equal to 55 per cent, of white tissue and 45 per cent, of grey
tissue in the entire l)rain.
About two years ago I had the happiness of conversing on this
matter with the late Mr. C. W. Merrifield, a gentleman of great
scientific attainments, and of clear and powerful mathematical
intellect, whose recent death the State, the scientific community,
his family, and his personal friends have great reason to deplore.
He took an immediate interest in the question, and embodied
the result of his deliberations on it in a memorandum, which I am
glad to be able to record in this place. He wrote to me :
' Your joroblem is exactly the same as that of the estimation of
gold in auriferous quartz. If for gold you read white cerebral
tissue, and for silica, grey matter, the formula solves your
problem.
i.
QUA NT AT ION OF CONSTITUENTS.
241
' Let g be the specific gravity of the heavier substance (gold) ;
„ (£ „ „ „ lighter „ (quartz):
„ m „ „ „ mixture;
a; be the unknown proportionate hulk of gold to a unit of
bulk of the mixture.
Then xg is the proportionate weight oi gold, and {l—x)q is the
proportionate weight of quartz. The sum of these must give the
weight of the mixture, that is —
xg-\-(l—x)q=mj
or x(g — q) = m- q,
m - q
or x =
g-'i
which gives the proportionate hulk of the gold.
' The proportionate weight of the gold is :
qx q m — q
or — ^.
m m g —q
Then, if ^ = 19, and ^ = 2-6, while 7?z = 9, we have m-^=6-4,
q-q = \Q'i.
. 6-4
Proportionate bulk '^fg^^^^^'
Proportionate weight =-^A x 1? = 0'824.
16 "4 9
' This method gives very good results when, as in the case of
auriferous quartz, the specific gravities are widely different,
because then a small error in the estimations of the data does
not make a large error in the result. It is otherwise when the
difference of density is small. For instance, suj)pose — •
g = specific gravity white tissue =1-035
2 = „ „ grey „ =1-025
m= ,, ,, brain altogether =1-031.
Then we have for proportionate hulk of white tissue :
m-g_0-006_Q.g ,
g-q 0-010 '
for proportionate iveighf of white tissue :
m g-q 1-031
16
r
242 CHEMICAL CONSTITUTION OF THE BRAIN.
' Now suppose the above data to be in error, so that in reality :
(/ = 1-034, instead of 1-035
^7= 1-024, „ 1-025
m being as before. Then -we have for
Bulk ^1^=^ = 0-7;
g-q 0-010
Weight I- = 0 -7 X = 0-70204,
m g-q 1-031
which is a very different result, an error of 1 per cent, in the two
specific gravities, and both in the same direction, altering the
result in the ratio of 6 : 7.'
Another method for the quantation of grey and white matter,
recently proposed, is based upon the difference in the quantity of
water contained in the two tissues, and expelled at 95°. We
have seen that grey tissue loses 85-27 per cent, while white tissue
70-23 per cent, of water. Now, if the loss of water of the entire
brain be known, and the foregoing data be physiological constants
or specially ascertained in each case, the relative weights of white
and grey tissue may be calculated.
D. SKETCH OF A SYSTEMATIC QUANTITATIVE ANALYSIS
OF THE BRAIN.
The brain is weighed in its membranes ; the latter are then
carefully removed and weighed, and their weight is deducted
from the weight of the brain previousl}^ found. The membranes
of a human brain will be found to weigh about 60 g. The brain-
tissue is then cut, or minced in a machine, and steeped in alcohol
until it has become hard. It is then worked through a sieve in
the manner described in the second chapter of this treatise. The
brain-pulp is now exhausted with sj^irit of 85 per cent, strength.
The boiling with spirit must be repeated until a litre of spirit
boiled with the whole of the residue, filteiT-d and distilled to dry-
ness, leaves only an inappreciable residue. The first spirit-extracts
which deposit the particular white matter are kept separate. All
particles of brain-tissue on the one hand and all portions of liquid
on the other must be constantly collected with scrupulous care. A
human brain will leave from 100 to 120 g. of dry albuminous matter.
The alcoholic solutions of the first extractions deposit on cool-
ing and standing the ichite matter, which amounts to 4-7 to 5 per
QUANTATION OF CONSTITUENTS.
243
cent, of the mixed brain-tissues, but is mainly derived from the
white tissue. (Compare on this subject the special analyses of
white and grey tissue given in the previous chapter.)
Analysis of the White Matter. — It is exhausted with ether, by
being shaken with it in a stoppered cylinder, and allowed to
settle ; the ether is drawn off with a syphon worked by air-pres-
sure. The white cerebrin mixture, containing the cerebrin and
some phosphorised matters, remains insoluble, while all kephalin,
lecithin, some myelin, all cholesterin, and some other neutral
matters, an oil or ether (cerebrol) and a yellow coloured matter,
dissolve. Thus we have :
Soluble
in ether.
Kephalin, part combined -
Lecithin, ,,
Myelin,
Cholesterin
Neutral (new) matter
Cerebrol
Yellow coloured matter -
All extracted.
All „
Part
All „
Part ., (Phrenosterin).
All ;,
All .„
Treatment of the Ether Solution ; separation of its Ingredients from
each other. — The ether is distilled off, the liquid residue is mixed
with an alcoholic solution of acetate of lead and excess of alcohol,
and boiled for some time under a reflux cooler. It is then allowed
to cool and stand. The cholesterin crystallises in the upper layer
of the mixture, while the kephalin lead and myelin lead remain
below, insoluble. The mixture is gently warmed until cholesterin
is dissolved, and again allowed to cool. This process is repeated
to cause the kephalin lead and the myelin lead to become as ad-
herent and lumpy as possible, so that when the warm spirit solu-
tion is filtered off through a filter on a hot funnel, only a minimum
of the insoluble compounds may pass on to the filter. The residue
is then exhausted with spirit by repeated long boiling with it.
Thus we have :
Insoluble in
boiling spirit.
Soluble in
boiling spirit
part.
Kephalin lead
Myelin lead
Cholesterin
Myelin lead
Lecithin -
Cerebrol -
All precipitated.
Greater part precipitated.
All.
Part.
All.
All.
I Yellow coloured matter All.
i Neutral new matter - All.
(Phrenosterin).
16—2
2U CHEMICAL CONSTITUTION OF THE BRAIN.
Treatment of the insoluble in boiling Spirit part (of the Ether Extract
boiled ivith Lead Acetate). — This contains all kephalin lead, and
some 'myelin lead, which have to be separated. This is done by
absolute ether, in which kephalin lead dissolves with a red colour,
while Mi/elin lead remains as an insoluble white deposit. The latter
is Avashed with ether by decantation mainly (with the aid of a
syphon and air-pressure), lastly on the filter, dried and weighed.
The datum is myelin lead part the first. The kephalin lead solution
and all ether used for washing myelin lead is distilled to dryness
and the residue weighed. It constitutes kephalin lead, and con-
tains all the kephalin which had been present in the white
matter.
In each, myelin lead and kephalin lead, the metal and phos-
phorus are ascertained by analysis, and from the data the amounts
of pure myelin and pure kephalin relatively are calculated.
Treatment of the soluble in Spirit part. — The boiling solution is
mixed with as much boiling water as it will bear without be-
coming precipitated, and is allowed to cool slowly. Cholesterin
(and phrenosterin crystaUises almost completely somewhat
later, and covering the latter settles the myelin lead. Lecithin
(? lead), cerebrol, yellow-coloured matter, neutral new matters
remain in solution. (This mixture requires further study.)
Separation of Cholesterin from Myelin Lead. — The isolated crystal-
lised matter is pressed, dried, and placed in absolute ether in a tall
stoppered cylinder, and frequently agitated. Cholesterin dis-
solves, while the lead salt of myelin and a cerebroside settle as a
white deposit. The extraction of cholesterin is completed by the
repeated application of large volumes of ether. The united ether
solutions are distilled to dryness ; the residue is dissolved in boil-
ing dilute spirit (if not sufficiently dilute, the boiling solution
must be mixed with boiling water until it becomes turbid), and
the solution set to crystallise. The cholesterin is filtered off,
dried, and weighed. The white salt which the ether dissolving
the cholesterin has left insoluble, is myelin lead, and a cere-
broside. This is dried and weighed. It gives the purple reaction
with oil of vitriol alone on standing, immediately with sugar-
syrup.
The spirit mother-liquor of the cholesterin, after concentration,
may yet yield a minute quantity of cholesterin, but does not con-
tain much else, colouring-matter excepted. It may be added to
QUANTATION OF CONSTITUENTS.
245
the principal mother-liquor, from which cholesterin and myelin
lead were originally deposited.
Solution in Spirit of Lecithin, Cerebrol, Yelloiv Colouring Matter,
Neutral Matter, and some Cholesterin. — Lecithin does not remain
combined with lead in watery spirit. It cannot be separated
from the rest of cholesterin except by precipitant reagents, or
by chemolysis, by which it is decomposed. Its quantation has
been found to be effected with the greatest approximation to
truth by the following process : The spirit solution containing
the matter just named is heated until all the spirit is evaporated.
The residue is then treated with boiling water, in which it hardens
and becomes granular. (In cold water it swells and becomes
pasty, so that it cannot be separated from its mother-liquor.)
The hot water is now decanted, and replaced by new ; the mix-
ture is allowed to ' cool, heated again to make the solids curdle,
and the water is again decanted. This is repeated until the de-
canted water is free from lead.
Chemolysis of the Lecithi7i, etc., Mixture uiih Barita. — The mixture
as described is now mixed with the necessary quantity of barita
hydrate and water, and chemolysed in a closed platinum tube
under pressure at 125° for at least six hours. The contents of
the tube are extracted firstly with hot water, which removes
excess barita, ghjcerophosphate of harium and neurin. The solution
is neutralised with carbonic acid, and the filtrate is evaporated.
When suitably concentrated, the addition of absolute alcohol to
it precipitates all harium glycerophosphate, while the filtrate con-
tains all neurin. The solution is neutralised by hydrochloric acid
in excess, and the addition to it of alcoholic platinic chloride
precipitates all neurin as platino-chloride hydrochlorate. Both
salts, the barium glycerophosphate and neurin double salt, are
dried and weighed.
Products of the Barita Chemolysis of the Lecithin Mixture which
are LnsoluUe in Water. — These are extracted with boiling spirit.
The concentrated spirit solution deposits yet some cholestsrin
and a small quantity of a barium salt. These are removed by
the filter. The cholesterin is separated from the barium salt
by ether. The spirit solution now retains the bodies above
mentioned — namely, two neutral crystallised bodies (which are
here noticed for the first time), and perhaps some cerebrol and
yellow colouring-matter. The two new bodies crystallise out of
246 CHEMICAL CONSTITUTION OF THE BRAIN
the absolute alcohol solution. Ether sej^arates the second crys-
tallised body from the first; the latter is recrystallised from
spirit. A thick mother-liquor remains, which requires further
qualitative examination. It is dried and weighed, and placed in
the account as last ]^)roduct of chemolysis of lecithin mixture.
That part of the product of the chemolysis of the lecithin
mixture which is insoluble in water as well as boiling spirit
contains the hariim salts of the fatty acids produced from the
chemolysed lecithin, as well as from the chemolysed ethylic ethers
of fatty acids resulting from a previous partial decomposition of
lecithin under the influence of heat and alcohol only. As they
contain barium carbonate, they have to be reconstituted in a pure
state, and the diff'erent fatty acids have then to be separated.
This is best done by decomposing the salts in water with hydro-
chloric acid, and extracting the fatty acids with ether. The ether
is distilled off, the fatty acids are dissolved in watery ammonia,
and precipitated as lead salts by lead acetate. The lead salts are
dried, powdered, and exhausted with ether. The ether solution
on distillation leaves lead oleate, while the salt insoluble in ether
will be found to be mainly lead imlmitate or mar gar ate , with only
little, if any, stearate. From the oleate and palmitate, with the
aid of the neurin and glycerophosphate, the amount of lecithin
originally present is easily calculated.
Treatment of the Alcoholic Solution which has deposited the White
Matter. — This solution, and all alcoholic extracts of the albu-
minous part obtained until it is exhausted, are distilled together
to a convenient state of concentration, and allowed to cool. A
matter is then deposited which, from its consistency, has been
called ' huttery, and which consists of cholesterin, lecithin, kephalin,
myelin, and some other ingredients. This is separated from the
liquid by filtration, and analysed as will be described.
Treatment of the Concentrated Alcoholic Solution ivhich has deposited
the Buttery Matter. — This solution is evaporated on the water-bath
until all alcohol has disappeared. The last portions of phos-
phorised matters and cholesterin, together with small quantities
of oily ethers, then float on the watery liquid and adhere to the
cvaporating-dish. This product is termed the Uast oily.' It is
most advisable to separate this from the watery solution without
the employment of a filter, and after slightly rinsing with dis-
tilled water, to add it to the Inittery matter for further analysis.
QUANTATION OF CONSTITUENTS.
247
Anahjsis of the United Buttery and Last Oily Matters. — To these
matters, after they have been dissolved in a sufficiency of hot
alcohol, the lead process, as described in the paragraph relating
to the ether extract of the white matter, may at once be api)lied.
Kephalin lead and myelin lead are precipitated ; lecithin, cho-
lesterin, and some myelin lead remain in solution, together with
other matters to be described. Thus we have —
Precipitated from and insoluble J Kephalin lead (all).
The hephalin lead, and myelin lead are separated from each other
by ether as above described, and their quantities weighed. The
lead contained in the respective preparations is ascertained by a
special quantation of the phosphorus and the lead, and from these
data the actual amount of each of the free phosphorised principles
is calculated.
The myelin lead and cholesterin deposited from the hot alcohol
are also separated by ether, and the products weighed. The
cholesterin may be weighed as residue from the ether solution
after distillation of the ether from a tared flask, or it may be re-
crystallised from very dilute spirit, and weighed in the crystallised
state. AVhen thus recrystallised, it is so pure that its melting-
point is mostly at 145°.
The cold spirit sohdion, containing lecithin, cerebrol, yellow
matter, neutral new matter, and mostly a residue of cholesterin,
together with some lead acetate, has now to be chemolysed, so
that its ingredients can be ascertained from the products of de-
composition. The spirit is first evaporated, and the residue
heated with water, which dissolves the acetate of lead and any
impurity soluble in water. This purification with hot water (cold
water has to be avoided, as it makes the residue swell and pre-
sent a semi-mucilaginous state) is repeated until the water is free
from lead. The residue is now treated as follows :
Chemolysls of the Last Residue of the Buttery and Last Oily Matter
in boiling spirit -
Soluble in boiling spirit, part
deposited on cooling
Cholesterin j deposi
Myelin lead >■ on
and a cerebroside I cooling.
Lecithin.
Cerebrol.
Yellow matter.
Neutral matters.
248 CHEMICAL CONSTITUTION OF THE BRAIN
2uhicJi ivas not precipitated hy Lead Acetate. — The necessity for this
process arises from the fact that the bulk of the lecithin cannot
be separated from the last traces of cholesterin and from the
small quantities of ether formed during the long processes
with alcohol. There are, moreover, matters present, such as
the neutral new matters, which are all soluble in ether as well
as alcohol, and do not combine with reagents in such a manner
as to become insoluble while the other bodies remain soluble, or
inversely.
The mixture as described is chemolysed in the same manner as
the residual mixture from the lead-precipitates from the ether-
extract of white matter described above. It is mixed with the
necessary quantity of barita hydrate and water, and heated in a
closed platinum-tube under pressure at 1^5° for at least six hours.
It may also be boiled in an open platinum dish, with frequent re-
newal of the water, for at least twelve hours ; but the process is
liable not to be complete, as the fatty acid salts formed may
enclose portions of lecithin and protect them from the influence
of the barita. Smaller quantities of matter may be enclosed in
glass tubes and sealed, and then heated to 125°, surrounded by
water in a closed brass tube. This last precaution is necessitated
by the fragility of the glass, if unprotected. The employment of
the platinum tube is preferable, owing to its simplicity. The
contents of the tube are extracted with hot water, which removes
excess of baiita, ghjceropJiosj^hate of barium and neurin. These are
isolated as above described — the glycerophosphate by alcohol,
and the neurin by platinic chloride.
The remaining solid products of the chemolysis, which are in-
soluble in water, are extracted with boiling spirit. In this all
cholesterin, and other matters not yet fully identified, and a
little barium salt of a fatty acid dissolve, while the barium salt
of the fatty acids produced by the chemolysis from lecithin and
cerebrol remain insoluble.
The spirit solution, when sufficiently watery, deposits all cho-
lesterin, which is dried and weighed ; it retains in solution the
neutral matters alluded to. This last mother-liquor is evaporated
to dryness and weighed, and the product is entered into the
record of the analysis as crystallisable undetermined products.
The harium scdls of the fatty acids, oleate and margarate or pal-
mitate, are decomposed with hydrochloric acid and water under
QUANTATION OF CONSTITUENTS.
249
ether, and the h'beratecl fatty acids are then combined, first with
ammonia, next with lead. From the dry mixture of lead oleate
and margarate the former is extracted by ether. The margarate
remains as a white substance, which is easily weighed ; the oUate
is best weighed as residue of the ether solution distilled from a
tared flask.
In calculating the amount of lecithin from the oleic and margaric
acids obtained as lead-salts, and comparing these data with those
obtained by calculation from the quantities of glycerophosphoric
acid and neurin, the following circumstances have to be borne in
mind :
Of the lecithin present in the brain, and extracted by the
alcohol, a part is already decomposed during the chemical opera-
tion. AYe shall see below how an excellent (juantatloii of the
lecithin can be made upon any portion of brain, provided it is not
intended to estimate many other or all the ingredients of the
extract obtained therefrom. But in the course of a complete
analysis of a single brain, such as is here described, the complete
quantation of the lecithin as such is not feasible, on account of
the decomposition just alluded to. This decomposition causes a
loss of neurin and glycerophosphoric acid on the one hand, which
remains in the watery mother-liquor containing the principles of
the brain soluble in water, and a loss of fatty acids on the other,
which combine with alcohol and form ethers. These ethers are,
of course, again decomposed during the barita chemolysis, so that
ultimately the whole of the fatty acids which were present in the
form of lecithin are obtained as barium salts. It follows, there-
fore, that when the quantities of fatty acids found are compared
with the quantities of neurin and glycerophosphoric acid, equiva-
lent for equivalent, there will be an excess of fatty acid over the
neurin and glycerophosjDhoric acid. It is therefore necessary to
take the fatty acids as the basis of calculation for the amounts of
lecithin present in the original extract, and the neurin and
glycerophosphoric acid only as subsidiary aids for the determina-
tion of the minimum and the control against accident.
Separation of the Ingredients of the Buttery Matter by a Process in
which Caustic Barita is em^jloyed. — In this process barita takes the
place of lead which is employed in the process just described.
The results are in the main analogous. The mother-liquors will
have to be chemolysed with more barita, as in the previous case,
250 CHEMICAL CONSTITUTION OF THE BRAIN
but the process will not be delayed by the necessity of removing
the excess of lead acetate.
The buttery matter is dissolved in a sufficient quantity of hot
spirit, and filtered hot. To the hot solution, hot barita water
(saturated in the cold) is now added, while the mixture is kept
boiling. A precipitate separates and becomes adhesive. The
solution is decanted and filtered boiling. The precipitate which
remains insoluble is exhausted with boiling spirit, dried and
treated with ether. KephaUn-harium goes into solution, while a
white salt {myelin-barium and small quantities of barium salts of
fatty acids) remains insoluble. The hot spirit solution, on cooling,
deposits a white precipitate, and then is almost free from ingre-
dients. The precipitate is isolated, dried, and exhausted with
ether. This solution of ether contains all cholesterin, and a
mere vestige of kephalin. The white precipitate contains, firstly,
a body soluble in boiling spirit, and deposited from it in needles
(curved needle body), and a body which is now, after removal
of the bodies soluble in ether, insoluble in boiling spirit, and
contains much barium and phosphorus.
The kephalin-hamm as precipitated from human buttery by
barita-water in the abov^e process is not yet a pure compound, as
was shown by the following tests of a specimen. It was insoluble
in boiling spirit, easily soluble in ether, twice precipitated by
absolute alcohol, and dried over oil of vitriol at 70°. It contained
2.3 '30 per cent. Ba. and 4 67 per cent. P.
The amount of barium found corresponds approximately to a
dibarium kephalin, which requires 24-77 per cent. Ba. But the
amount of phosphorus is in excess of that theory, which requires
2 '9 per cent. P, and must be left unexplained. Dibarium-kephalin
has an analogue in a diplumbic kephalin, which I have described
in the chapter relating to kephalin.
Analysis of the Cerehrin Mature. — This mixture consists of a
number of well-defined immediate principles, which belong mainly
to three distinct categories.
(1.) Cerehrositles or bodies of the glucoside type, which contain
as constitutional base a peculiar sugar, cerehrose. The type of
these bodies, pthrenosin, forms the main quantity of the ingredients
of the mixture. It is insoluble in cold absolute alcohol. Keratin
is soluble in much cold absolute alcohol, at least for some time,
and is deposited slowly on standing in a semi-crystalline form.
QUA NT AT ION OF CONSTITUENTS.
251
A body crystallising apparently in curved needles, bregenin, is
permanently soluble in alcohol. These bodies do not combine
with lead when it is added as acetate to their solution in spirit.
But there are a number of cerebrosides which do combine with
lead when it is added as acetate to their spirit solution : of this
class is cerebrinic acid, and spherocerehrin, and the three bodies
accompanying spherocerebrin as described in the article on the
cerebrinacides.
(2.) Fhosphorised bodies, which, owing to some of the fatty
acids contained in them being identical with or nearly allied to
the fatty acids contained in the cerebrosides, have the same or
very nearly the same solubility in alcohol and other solvents as
the cerebrosides, and therefore follow them pertinaciously :
sjphingomyelin and apomyelin.
(3.) Sulphurised bodies, of which a preliminary description has
been given in a previous chapter.
The cerebrin-mixture also always contains varying quantities of
bases, particularly potash and soda.
The cerebrosides being mostly neutral bodies, having no affinity
for either acid or alkali, can be isolated with solvents only. Of
the cerebrin-mixture an elementary analysis should be made, and
its results stated in atoms w^ith sulphur as unit, and again with
phosphorus as unit. This will at once show the proportions of
atoms to each other, and be the chief control of the processes of
quantitative separation to be undertaken afterwards.
The cerebrin mixture, dissolved in alcohol, may then be treated
with lead acetate and a little ammonia, and^he precipitate may
be exhausted with boiling spirit. This process separates the
mixture of cerebrosides into the two categories described above
under (1). The sulphurised bodies remain principally in the
lead precipitate, the phosphorised principles distribute themselves
over precipitate and solution. The separation and quantation of
these matters requires further study.
The nitrogen in the cerebrosides is probably all present in a
form, which by chemolysis with barita or sulphuric acid, suffi-
ciently long continued, will appear as sphingosin. Thus neurin
on the one, and sphingosin on the other hand, will probably be
the only nitrogenised nuclei to be isolated by chemolysis. They
are certainly the principal ones as regards quantity ; should the
sulphurised bodies contain any nitrogen and that in a particular
252 CHEMICAL CONSTITUTION OF THE BRAIN.
form, then the quantity of this particular product would be much
below the quantities of the products just mentioned.
E. A PRELIMINARY EXPERIMENT FOR THE QUANTATION OF
CONSTITUENTS OF AN ENTIRE HUMAN BRAIN.
Of the left hemisj^here, which when entire weighed 596 g.,
460 g. were taken for the following quantations. The tissue was
comminuted and exhausted with boiling spirit, etc., and yielded
the following educts :
Albuminous matters =35 '06 g., equal to 7-62 per cent, of
tissue.
JVhite matter, deposited from spirit, 21*93; the same after ex-
traction with cold ether (cerebrin-mixture) dry = 12 "28 g. Soluble
in ether, 9*65 g. These cerebrosides, etc., boiled with absolute
alcohol, gave —
(a.) Less soluble cerebrins, phrenosin, etc., deposited imme-
diately 8-11 g.
(b.) More soluble ones, kerasin, etc., deposited after days 0*56 g.
And left insoluble stearoconote, albumen, and paper fibres 0'78 g.
Of this last item 0*53 were soluble, 0*25 insoluble in hot benzol.
The insoluble in benzol part contained a body which left a black
ash on combustion, and contained much phosphorus. It was an
earthy compound of a phosphorised body. It gave a brownish
red, but no genuine oleo-cholide reaction.
The alcoholic solution from the cerebrins = 340 cc. was divided in
two equal halves of 170 cc. each. To one half platinic chloride
was added as long as a precipitate was produced. The precipitate,
containing a phosphorised body, weighed 0*85 g., and contained
9-58 per cent. Pt ; it yielded further 3*13 per cent. P. To the
second half of the alcohol solution cadmic chloride was added,
and the precipitate obtained weighed 0-8124 g. ; it yielded
13*27 per cent. Cd and 3*42 per cent. P.
Of the matters dissolved in spirit, and not precipitated by these
alkaloid reagents, 0*3619 g. were yet precipitated by water.
The ether-extract from the cerebrln mixture weighed 9*65 g.
The butter// matter, soluble entirely in cold ether =21*65 g.
The last oihj matter, treated with lead acetate, yielded lead pre-
cipitates which together weighed 11 '8450 g.
Out of these there was obtained lecithin =2 8995 g. (This was
QUA NT AT ION OF CONSTITUENTS.
253
easily soluble in cold absolute alcohol, and gave the characteristic
tests with platinic and cadmic chloride.)
Further kejjhalin lead = 0-8435 g. = 0*5659 g. keplialin ; and
myelin lead = 6*6080 g. =5*2041 myelin.
The last watery extract gave a phosphomolybdate precipitate of
alkaloids, which weighed dry 3*7084 g. It yielded further 1 *56 g.
zinc lactate dried in vacuo over oil of vitriol =1*0074 g. pure
sarcolactic acid.
The inosite amounted to 2*5335 g.
The undefined organic extractives weighed 2*7822 g.
The scdts, as carbonates, weighed 1*7218 g. Of these 0*39 were
potassium =0*745 KCl, and 0-39 sodium =0*9844 NaCl. The
salts as chlorides weighed 1*7294 g., and out of this mixture
2-4610 g. PtCl4(KCl)2 were obtained.
In the calculation of kephalin, myelin, and lactic acid, the
following formulae were used, which should be considered as
provisional, and will have to be tested by further, or supple-
mented by direct analysis of the products :
Kephalin, rnolec. weight = 836 ; kephalin lead =C42H^5NPOj3Pb2
M.W. =1246.
Myelin, M. W. = 760 ; myelin lead = C^oH.gPbNPOio, M. W. = 965.
Sarcolactic acid CgHgOg, M.W. = 90.
Zinc sarcolactate dihydrate, CqE^qZuO^ + 2B.fi, M.W. = 279.
The buttery matter which had been soluble in ether (21*6450 g.)
was boiled with barita and yielded 6*7 g. of cholesterin, .^ith
16*5 g. of barium salts of fatty acids. . The latter contained oleate
(as shown by the oleo-cholide reaction), much phosphorus, and a
small quantity of a body which was soluble in ether. But the
complete extraction with ether was impracticable, as the turbid
liquid would neither settle nor allow itself to be filtered. This
difficulty is not rarely met with when dry barium or lead salts
of brain educts are subjected to ether treatment for the extraction
of some ingredient.
The ether extract from the cerebrin mixture, containing the
kephalin and cholesterin, was treated with alcoholic lead acetate
and filtered boiling. The insoluble residue of kephalin lead
weighed 2*6410 g., which, assumed to contain 2Pb, is equal to
1772 g. free kephalin.
The solution in spirit of the cholesterin, etc., was evaporated, and
the residue freed from lead acetate by hot water. The cholesterin
254 CHEMICAL CONSTITUTION OF THE BRAIN,
was then re-crystallised and weighed. When the cholesterin thus
obtained was re-dissolved in ether, a white matter remained in-
soluble, weighing 0-2680 g., and being entirely combustible on
platinum foil.
Quantation of the Ingredients of the Eight Hemisphere.
The total hemisphere^ with the membranes, weighed 589 g. ;
after removal of membranes, 564 g. ; of this quantity, 465 g. were
employed in the following quantations :
The albuminous substance amounted to 35-68 g., equal to 7-66
per cent, of the hemisphere tissue.
The ivhite matter deposited from spirit dry was = 18'60 g.
The ether-extract from this 6-97 g.
The cerebrins insoluble in ether 11-63 g.
The buttery matter (= 21-66 g.) in alcohol was treated with lead
acetate, and boiled. The precipitates insoluble in boiling alcohol
were suspended in ether for separation, but the cylinder containing
the mixture breaking spontaneously, the quantation was lost. In
the table this void is filled up by data obtained from the data
concerning the left hemisphere by calculation.
Tlie inosite was obtained in two portions, one with neutral
acetate (it was not previously known that inosite was so preci-
pitated), another with basic, together about 0*43 g. The lead
having been removed from the liquid with sulphuric acid, the
lactic acid was extracted ^and formed into zinc salt. It weighed,
dry at 100°, 0*87 g. =0-64 sarkolactic acid.
Alkaloids were precipitated from the mother-liquor by phospho-
molybdic acid. The precipitate Aveighed 3 8 g., and contained
1 -30 g. mixed alkaloids.
The mother-liquor was treated with barita, etc., evaporated,
and the residue burnt. The indefinite extractives amounted to
2-63 g.
Of lootassium 0-25 g., of sodium 0*40 g., were obtained.
The hot mother-liquor of the buttery matter treated with lead
acetate (of which operation the insoluble precipitates were lost as
above described), on cooling deposited myelin lead and cholesterin,
weighing, when dry, 6-99 g. The myelin weighed 5*22 g.
The spirit filtrate, containing lecithin and little cholesterin, after
evaporation, left a residue weighing 8*13 g.
The remaining bodies containing cholesterin were chemolysed
QUANTATION OF CONSTITUENTS.
255
with barita, and gave cJioIesterin = 8-93 g. pure, and 8-80 g. harkim
salts of fatty acids.
Quantation of the Ingredients of the Cerehellum.
The total weight of the fresh cerebellum with membranes was
135 g.j and after removal of membranes, etc., 124 g. were analysed.
The albuminous matter amounted to 11-3809 g. = 9-17 per cent,
of cerebellum.
The ivhite matter from first spirit was = 1 "81 g. Of this, 0*2185 g.
were soluble, 1*6645 g. insoluble in ether =cerebrins.
The filtrate from the ivhite matter was evaporated and treated
directly with lead acetate. There were obtained kephalin lead
= 1*97 g. ; myelin lead 1*65 g. ; in the solution, cholesterin
with lecithin and myelin lead =3*26 g. j a second portion of
myelin lead with lead salt insoluble in ether containing inosite
= 0*05 g. This inosite having been precipitated by neutral lead
acetate (a reaction hitherto unknown) made the quantation of
this body inaccurate. It amounted probably to 0*66 g. for the
entire cerebellum.
The lactate of zinc, dry at 100°, weighed 019, equal to 0*1352 g.
sarkolactic acid.
21ie alkaloids, precipitated by phosphomolybdic acid, weighed in
combination 1-62 g., free 0*6920 g.
The extractives, mixed with the salts, weighed 1*55 g.
The alkali salts consisted of 0*01 potassium and 0*02 sodium.
The mixture of cholesterin, lecithin. and myelin lead, above de-
scribed as weighing 3*26 g., was boiled with barita and a little
spirit for three hours. The watery solution of neurin glycero-
phosphate and excess of barium was separated from the insoluble
matter. The cholesterin was extracted by boiling spirit, and
weighed 1*95 g. Its melting-point was 145°. The barium scdts of
the fatty acids weighed 2-56 g.
Quantation of the Constituents of the Mesenkephalon.
The mesenkephalon and medulla oblongata weighed 34 g.,
without the membranes 33 g.
The albumen amounted to 2*48 g. = 7*5 per cent, of the mesen-
kephalon.
The luhite matter weighed 0*64 g. ; of this 0*03 were soluble in
ether, 0-56 insoluble.
256 CHEMICAL CONSTITUTION OF THE BRAIN.
The huttery matter yielded 0*67 kephalin lead.
The lactate of zinc weighed 0-11, air dry, equal to 0*07 lactic acid.
The rest of the matters soluble in water were not estimated on
account of their small quantity.
The data thus far ascertained have been arranged in the follow-
ing table. Thej^ claim to be minima only, and with improved
processes somewhat larger quantities will probably be found.
Blanks are left where the quantations could either not be made,
or were unsatisfactory when the products were tested.
The experience gained by this analysis has shown that the
division of all the educts of a brain into five primary categories
is practical. They are (1) albumen, (2) white matter, (3) buttery
matter, (4) last oily matter, (5) matters soluble in water. Of
these, the last oily and the buttery matters maj^ be treated
together for the separation of their ingredients, when the cere-
bellum or mesenkephalon are concerned. When derived from
the hemispheres these products are more conveniently kept apart.
The table contains about 130 data. But it will be seen that
the cerebrosides, e.g., occupy only one column (col, 6), whereas
probably ten columns will be required to register the quantities of
various specific bodies of which the insoluble in ether part of the
white matter is composed. I estimate, therefore, that the quanti-
tative analysis of one brain will involve the production and
weighing of about 300 definite bodies or compounds. Each of
the four divisions of the brain, and each of the two varieties of
tissue, the white and the grey, would thus require about fifty
quantations for chemical characterisation.
The loss registered in the table appears at first sight enormous.
One part was incurred by diffusion from the parts immersed
in water for the estimation of the specific gravities as described
in a previous chapter. This quantity was 1-65 g., as ascertained
by eva2:)oration of the water and weighing of the dry residue. An
uncertain part of weight was lost by evaporation. But the
greatest part was lost in the course of the anatomical separation
of white and grey matter, in the course of comminution, and
transfer from filter to filter and vessel to vessel. This loss may
be much diminished by improved apparatus, but in the present
case it imports only a slight degree of inaccuracy into the general
result of the analysis, as its effect has been supplemented by a
proportional calculation.
QUANTATION OF CONSTITUENTS,
m
>
I— I
H
H
w
H
o
si
Sodium.
CO ;
OCiC-l OO-t^ OOi T-ll^
-tilOO OO OO OO
OOO OO OO OO
90-1
Potassium.
0-25
0-39
0-01
0-01
0-03
0-004
0-01
0-001
0-07
OO
1^
o
'O
Indefinite Extrac-
tives.
C0C0O5 CO-* COr-H -tl(M
CO ^."^ *? 'P
'f^JifjAn OO OO OO
o
p
Hypoxanthin and
Alkaloids.
O CO O I-H T-l
TO O CO 11 1 r-l P C^'
Ah O ' ' ' O O O
CO
CO
Lactic Acid.
^OCO l-«kO O-H OO
COOi-i OO OO 0(M
Ot-hO OO OO OO
rH
Ol
CO
Inosite.
CO 00 O CO -i^ O^ O QO
CO p O rH p --O p p
OOO OO OO OO
p
T— 1
Cholesterin Total.
COOilO 1— iiO OO CNCO
010505 Or— ( COOi >— 100
OO'-Oi-t i-H'M OO Oi-I
I-H
T— (
Lecithin Total.
O 1-^ OO W CO Ol o
O I O O 1^ O i-H
o'cN OO OO Or-I
o
Myelin Total.
CO
Ol O to coco OJfN OOS
OJO^l^ CMr-H OO OCO
lOiCCO OO OO OO
f— >
?
Kephalin Total.
CM
1 \a ^ o *0 CO
CO 1 <>i 7*^ p <? T' o p
CO I-H OO OO OO
p
00
Buttery Matter.
CO CO -h
CO p (>4 1 CO 'r* 1 IT'
I-H CO ' o o ' ' ^
'M (N
x>
CO
TV. ^vj., feDiuuie 1X1
Ether.
J^iOOI COfN COO -^lOO
CiCOOq O^ coco OOO
COOlO 0(M OO OrH
OO
tH
?o
W. M. insoluble in
Ether.
CO OO CO CO CO f -H
to to to I 1
Ah 0^ Ah O o 1 i
I-H I-H
lo
CO
CO
i U-S
vvnite iviatter.
OCOCO --+HOO l^O
COOOO COCi OlCfJ Oi-H
COi-HrH OCO OO O-H
1—1 (M
i
Albumen.
1
OOCO'* coo OO C050
CO o as -+11^ QO lo o
lO K-i O Ol O CO O CO
CO CO rH
1 o
!
Is
CO
Quantities
analysed.
OJ P
»OCO^ coco CSCO CO CO
:o CO (M CO CO ■ CO
1 -+I rH
1
1 ^
OI
Weights without
Membranes.
^ O Oi CO
CO lOI CO 1 1 1 ' 1 1
>0 O I-H 1 II II
CO
Oi
CM
Weights with
Membranes.
1 as CO m -H
1 CO Oi CO 00 1 j 1 II
>!0
CO
S » Ph
17
258 CHEMICAL CONSTITUTION OF THE BRAIN.
I have no doubt that by continued stud}^ the quantitative
analysis of the brain may attain a very high degree of accuracy.
This belief is based upon a number of compounds and processes
described in the chapters relating to the different principles, which
may here be summarily referred to, although there has not yet
been time for giving them places in a systematic analytical
process.
The kejjhallns may be combined with lead, barita, or cadmium
chloride. All these compounds are soluble in ether, insoluble in
alcohol.
The lecithins may be combined with cadmium chloride. These
compounds are insoluble in ether, insoluble in cold alcohol,
soluble in boiling alcohol, soluble in cold benzol.
The myelins must be combined with lead ; in that state they are
insoluble in alcohol and ether, hot or cold.
The paraimjelins may be combined with cadmium chloride ; in
that state they are soluble in hot benzol, insoluble in cold, in-
soluble in ether.
The amidomyelins may be combined with cadmium chloride ; in
that state they are insoluble in hot or cold benzol, and insoluble
in ether.
The spiling omyelins are, as cadmium chloride salts, soluble in
hot benzol, and soluble in much cold benzol ; benzol, therefore,
offers no facilities for their separation.
The assiirins are not precipitated from alcohol by either lead
acetate or cadmium chloride, but by platinic chloride.
Phrenosin and kerasin are soluble in boiling spirit, insoluble in
cold ; the later deposition of kerasin affords means for its sei)ara-
tion. They are insoluble in ether, and do not combine with lead,
or cadmium chloride.
Kriaosiii is soluble in hot ether, insoluble in cold ; soluble in
boiling alcohol.
Bregenin is soluble in cold ether and cold alcohol. Neither
krinosin nor bregenin combines with lead, or with cadmium
chloride.
The cerehrinacides combine with lead, and as lead compounds
are insoluble in boiling alcohol ; a part of the lead compounds is
.soluble, another insoluble in benzol.
Many of the phosphatides can also be combined, like assurin,
QUA NT A no N OF CONSTITUENTS.
259
with platinic chloride. If we add to these reagents the means
furnished by limited chemolysis, and by complete chemolysis, it
will be seen that the quantation of any one of the well defined
ingredients is now feasible. Bat I have no doubt that specific
solvents, as well as precipitants, will be found for all the brain
educts or their compounds. Thus a few trials with acetone have
shown that it will be usef td in the separation of the cerebrosides ;
in a similar manner chloroform will be an occasionally useful sol-
vent.
The power of mercuramin for the removal of all acids from
any solution, and their recovery from the precipitate, gives to
the analyst a power which was undreamed of a few years ago.
The power of phosphomolybdic and phosphotungstic acids for the
isolation of alkaloids has made their extraction amenable to pure
reagents. And we can see from the behaviour of many of the
educts with even commonplace reagents that amongst them
there are at least some which will furnish means for stoichiometric
proceedings.
When the normal composition of the brain shall be known to
the uttermost item, then pathology can begin its search for
abnormal compounds or derangements of quantities. Thus the
amyloid degeneration is specific to brain and nerve-tissue, and
can be considered hypothetically as a reduction of an ingredient
of decomj^osed cerebrosides. This hypothesis has the advantage
that it is as yet the only one which can be made regarding this
remarkable disease. I believe that the great diseases of the
brain and spine, such as general paralysis, acute and chronic
mania, melancholy, and others, will all be shown to be connected
with specific chemical changes in neuroplasm, the products of
which cannot be more complicated than the chemolytic products
of the educts ; they need, however, not be identical with
chemolytic products, but may be new morbid products. Here is
a field for inquiry of the possession of which the guardians of
refuges for the insane will hereafter, I have no doubt, endeavour
to make good use.
The knowledge of the composition and 23roperties of neuro-
l^lasm and of its constituents will also aid us in devising modes of
radical treatment in cases in which at present only tentative
symptomatic measures are taken. In short, it is probable that by
17—2
260 CHEMICAL COXSTITUTION OF THE BRAIN.
the aid of chemistry many derangements of the brain and mind,
which are at present obscure, will become accurately definable
and amenable to precise treatment, and what is now an object
of anxious empiricism will become one for the proud exercise of
exact science.
INDEX.
Acetic acid, 216.
Acids (as principles), 199.
^sthesin, 160.
Albuminous principles, 211.
Alcohols, 199.
Alkaloids, 192, 225.
Amido-acids, 196.
Amidokephalin, 104.
Amidolipotides, 188.
Amidomyelin, 98, 44.
„ Preparation of, 99.
,, Properties of, 102.
Analysis, Consideration of methods of,
139.
Apomyelin, 99.
Assurin, 120.
Bregenin, 188.
Buttery matter, 31.
Calcium acid glycero-phosphate, 79,
129.
Caramels, 148, 158, 166, 171, 179.
Carbohydrates, 199.
Cerebrinacides, 178.
Cerebrinic acid, 179.
Cerebrose, 143, 170.
Cerebrosic acid, 145.
Cerebrosides, 134, 250.
„ Separation of, 136.
Cerebrosulphatides, 185.
Chemolysis, Apparatus for, 141.
Cholesterin, 199.
„ Reactions of, 200.
„ Isomers of, 201.
Cholophosphatides, 124.
Cupric inosite, 202.
Cytophosphatides, 125.
Formic acid, 209.
(Ilycerophosphoric acid, 128, 9, 76.
Glycoleucin, 221.
(4rey tissue, 231.
Hematophosphatides, 124.
Hypoxanthin, 192.
Imides, 196.
Inorganic bases, 1 26.
Inorganic principles, 229.
Inosite, 202, 198.
Inosite copper, 202.
Isocholesterin, 201.
Istarin, 120.
Kephalic acid, 73.
Kephalin, 52.
,, Chemolysis of, 72.
„ Compounds of, 61.
„ Constitution of, 89,
„ Dialysis of, 55.
„ Preparation of, 52.
„ Reactions of, 59.
Solubility of, 58.
Kephaloidin, 67.
Kephalophosphoric acid, 73.
Kerasin, 172.
„ Chemolysis of, 177.
,, Preparation of, 172,
,, Reactions of, 175.
„ Solubility of, 174.
Krinosin, 191.
Lactic acid, 205.
Lactophosphatides, 123.
Last oily matter, 32.
Lecithin, 42.
„ Chemolysis of, 49.
„ Compounds of, 46.
Leucin, 196, 214,
Leucin copper, 217.
Mercuramin, 259.
Myelin, 94.
., Compounds of, 96.
„ Preparation of, 95.
Needle body, 184.
262
INDEX.
Neuiin, 76.
Neui-oplasiii, 1.
Neuroplastin, 212.
Neurostearic acid, 169.
Neurostearic ether, 162, 109.
Xitrogenised fats, 188.
Oleic acid, 51.
Oxigenised principles, 199.
Oxikephalin, 64.
Oxikephaloidin, 70.
Paracholesterin, 201,
Paramyelin, 91, 44.
„ Preparation of, 93.
Peroxikephalin, 66.
Pettenkofer's test, 156.
Phosphatide.^ 39.
Phosphorised principles, 39.
Phrenosin, 138, 164.
,, Chemolysis of, 141.
„ Constitution of, 168.
„ Preparation of, 138.
„ Reactions of, 167.
Phytosterin, 201.
Psychosin, 152, 154, 164, 170.
„ Chemolysis of, 155.
Quantitative relations, 231.
Quantation of constituents (of entire
brain), 252.
Raspail's reaction, 156.
Specific gravity of brain, 237.
Spherocerebrin, 183.
Sphingol, 116.
Sphingomyelic acid, 115.
Sphingomyelin, 105.
,, Chemolysis of, 115.
,, Compounds of, 118.
,, Preparation of, 108.
„ Properties of, 113.
Sphingosin, 149, 169.
„ Compounds of, 150.
Sphingostearic acid, 116.
Stearoconote, 23.
Succinic acid, 209.
Sulphurised principles, ISO.
Tyrosin, 196, 214, 216, 223.
Water of colloidation, 2.
White brain tissue, 234.
White matter, 30.
THE END.
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ABERCROMBIE (J.) On Tetany in Young Children 15
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BRAMWELL (Byrom) Diseases of the Spinal Cord 29
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BROWN (George) The Student's Case-book 14
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CANTLIE (Jas.) Atlas of the Hand 10
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CHAUVEAU-FLEMING. Comparative Anatomy of the Domestic Animals 36
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COCKLE (John), Contributions to Cardiac Pathology 21
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COHNHEIM (Prof.) On the Contagiousness of Consumption 16
COLE (A. C.) Studies in Microscopical Science 25
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ORMSBY (L. H.) Deformities of the Human Body 17
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OWEN (Lloyd) Translation of Giraud-Teulon's (Anomalies of Vision) 19
PALFREY (J.) Atlas of the Female Organs of Generation 10
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Bailliere, Tindall, and Cox's Books. 13
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16 Bailliere, Tindall, and Cox's Books.
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I
18 Bailliere, Tindall, and Cox's Books.
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Bailliere, Tindall, and Cox's Books. 19
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20 Bailliere, Tindall, and Cox's Books.
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Hair. The Hair : its Growth, Care, Diseases, and Treatment ; |
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1
Bailliere, Tindall, and Cox's Books. 21
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Bailliere, Tindall, and Cox's Books. 29
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30 Bailliere, Tindall, and Cox's Books
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